Skin substitutes with improved barrier function

ABSTRACT

The present invention relates to in vitro cultured skin substitutes, and in particular to in vitro cultured skin substitutes that have improved barrier function. In some embodiments, improved barrier function is a result of improved culture conditions, while in other embodiments, improved barrier function results from genetic modification of keratinocytes. Improved culture conditions to improve barrier function include organotypic culture in the presence of linoleic acid and/or linoleic acid at about 75% humidity. Suitable genetic modifications for improving barrier function includes transfection with a DNA construct capable of expressing GKLF.

This application is a divisional of U.S. application Ser. No. 10/087,346(allowed) filed on Mar. 1, 2002 now U.S. Pat. No. 6,974,697, which inturn claims priority to U.S. provisional patent application Ser. No.60/273,034, filed Mar. 2, 2001.

This patent application was supported in part by NIH SBIR grant number 1R43 AR47499-01. The government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to in vitro cultured skin substitutes, andin particular to in vitro cultured skin substitutes that have improvedbarrier function. In some embodiments, improved barrier function is aresult of improved culture conditions, while in other embodiments,improved barrier function results from genetic modification ofkeratinocytes.

BACKGROUND OF THE INVENTION

There is a large market for test products and services that can predicteffects of skin barrier function. There is a particular need forvalidated assays that would enable companies with early stage compoundsto test for barrier function without resorting to expensive animal andhuman patch testing. Cosmetic companies spend approximately $50 millionto $100 million per year on this type of testing. Household product andpharmaceutical companies make similar expenditures.

Furthermore, there is a substantial market for products for burn therapyand for the repair or support of appropriate epithelial tissues andother wound and skin closure uses. For example, venous leg ulcers affectabout 1 million people in the United States and 3 million worldwide, andother ulcer conditions such as diabetic ulcers and pressure ulcers(bedsores), affect approximately 10 million people worldwide. Venousulcer standard care can take over 6 months to heal a wound and cost inexcess of $10,000.00. Furthermore, foot ulcers are a leading cause ofhospitalization among diabetics and are estimated to cost the U.S.healthcare system over $1 billion annually. Estimates forhospitalizations for burns in the United States range from 60,000 to80,00 annually, and costs for recovery from acute injuries range from$36,000 to $117,000 per patient.

A major function of human skin is to provide a barrier to transcutaneouswater loss and a barrier to prevent entry of toxic compounds ormicroorganisms. Development of the epidermal permeability barrierrequires the coordinated synthesis and metabolism ofkeratinocyte-specific protein and lipid products that are assembled intothe outermost skin layer, the stratum corneum. The expression of many ofthe key enzymes required for synthesis of these extracellular lipids isup-regulated during keratinocyte differentiation or following disruptionof epidermal barrier function, suggesting the existence of transcriptionfactors that function to promote barrier function development (Sando etal., J. Biol. Chem., 271: 22044-51 (1996); Watanabe et al., J. Biol.Chem., 273(16): 9651-5 (1998)). Barrier function is impaired by exposureto irritating substances, by infection and by a number of diseasesincluding atopic dermatitis and psoriasis. Environmental stresses canexacerbate the effects of these conditions on the essential barrierfunction of the skin. Many industries are interested in what effecttheir product has on barrier function of the skin. For example,companies that deliver pharmaceuticals transdermally need to facilitatethe penetration of the active agent past the barrier. Cosmetic companiesare interested in finding formulations that improve the barrierfunction.

In order to test compounds or formulations early in the developmentprocess with speed and accuracy, it would be beneficial to have an invitro test system that mimics the barrier properties of human skin.However, published studies indicate that existing skin equivalentcultures, such as EPIDERM, SKINETHICS or EPISKIN, have very poor barrierfunction (Ponec et al., J. Invest. Dermatol., 109(3): 348-55 (1997)).There has been substantial recent progress, however, in understandingthe importance of vitamin C, nuclear hormone receptors, lipid synthesis,and humidity on the proper development of barrier function (Ponec etal., J. Invest. Dermatol., 109(3): 348-55 (1997); Denda et al., J.Invest. Dermatol., 111(5): 858-63 (1998); Hanley et al., J. Clin.Invest., 100(3): p. 705-12 (1997); Hanley et al., J. Invest. Dermatol.,113(5): 788-95 (1999)). In many cases, these studies focus on chemicalor environmental signals that trigger the natural developmental programthat establishes barrier function at a specific time in utero. Clearly,a great need exists for skin substitutes having improved barrierfunction.

SUMMARY OF THE INVENTION

The present invention relates to in vitro cultured skin substitutes, andin particular to in vitro cultured skin substitutes that have improvedbarrier function. In some embodiments, improved barrier function is aresult of improved culture conditions, while in other embodiments,improved barrier function results from genetic modification ofkeratinocytes.

The present invention provides compositions comprising a human skinequivalent, the skin equivalent having a surface electrical capacitanceof from about 40 to about 240 pF. In some preferred embodiments, theskin equivalent has a surface electrical capacitance of from about 80 toabout 120 pF. In other preferred embodiments, the combined content ofceramides 5, 6, and 7 in the skin equivalent is from about 20 to about50% of total ceramide content. In still other preferred embodiments, thecontent of ceramide 2 in the skin equivalent is from about 10 to about40% of total ceramide content. The present invention is not limited toskin equivalents formed from a particular source of keratinocytes.Indeed, the skin equivalents may be formed from a variety of primary andimmortal keratinocytes, including, but not limited to NIKS cells. Instill further embodiments, the keratinocytes express exogenous wild-typeor variant GKLF. In still further embodiments, the keratinocytes arederived from two or more different sources.

In other embodiments, the present invention provides isolatedkeratinocytes comprising a sequence encoding GKLF operably linked to anexogenous promoter. In still further embodiments, the present inventionprovides an organotypic culture keratinocytes comprising a sequenceencoding exogenous GKLF operably linked to an inducible exogenouspromoter.

In some embodiments, the present invention provides methods for makingskin equivalents having improved barrier function. In some embodiments,the methods comprise providing keratinocytes and a culture mediacomprising ascorbic acid and linoleic acid; and culturing thekeratinocytes under conditions such that a skin equivalent havingimproved barrier function is formed. In some embodiments, the cultureconditions include culture at about 50 to 95% humidity, preferably about75% humidity. In some preferred embodiments, the ascorbic acid isprovided at concentration of from about 10 to 100 micrograms/ml. Infurther preferred embodiments, farnesol is provided at a concentrationof from about 10 to 100 micromolar. In still further preferredembodiments, linoleic acid is provided at a concentration of from about5 to 80 micromolar. The present invention is not limited to skinequivalents formed from a particular source of keratinocytes. Indeed,the skin equivalents may be formed from a variety of primary andimmortal keratinocytes, including, but not limited to NIKS cells. Instill further embodiments, the keratinocytes express exogenous wild-typeor variant GKLF. In still further embodiments, the keratinocytes arederived from two different sources. In other embodiments, the skinequivalent has a surface electrical capacitance of from about 40 toabout 240 pF. In some preferred embodiments, the skin equivalent has asurface electrical capacitance of from about 80 to about 120 pF. Inother preferred embodiments, the content of ceramides 5, 6, and 7 in theskin equivalent is from about 20 to about 50% of total ceramide content.In still other preferred embodiments, the content of ceramide 2 in theskin equivalent is from about 10 to about 40% of total ceramide content.In still further embodiments, the present invention provides the skinequivalent made by the method just described.

In other embodiments, the present invention provides methods of makingskin equivalents having improved barrier function comprising: providingkeratinocytes and a DNA construct comprising a sequence encoding GKLFoperably linked to an exogenous promoter; transfecting the keratinocyteswith said DNA construct to provide transfected keratinocytes; andculturing the transfected keratinocytes under conditions such that askin equivalent having improved barrier function is formed. In someembodiments, the culturing step comprises culturing the transfectedkeratinocytes in a culture media comprising ascorbic acid and linoleicacid. In some preferred embodiments, the ascorbic acid is provided atconcentration of from about 10 to 100 micrograms/ml. In still furtherpreferred embodiments, linoleic acid is provided at a concentration offrom about 5 to 80 micromolar. The present invention is not limited toskin equivalents formed from a particular source of keratinocytes.Indeed, the skin equivalents may be formed from a variety of primary andimmortal keratinocytes, including, but not limited to NIKS cells. Instill further embodiments, the keratinocytes express wild-type orvariant GKLF. In still further embodiments, the keratinocytes arederived from two different sources. In other embodiments, the skinequivalent has a surface electrical capacitance of from about 40 toabout 240 pF. In some preferred embodiments, the skin equivalent has asurface electrical capacitance of from about 80 to about 120 pF. Inother preferred embodiments, the content of ceramides 5, 6, and 7 in theskin equivalent is from about 20 to about 50% of total ceramide content.In still other preferred embodiments, the content of ceramide 2 in theskin equivalent is from about 10 to about 40% of total ceramide content.In still further embodiments, the present invention provides the skinequivalent made by the method just described.

In still other embodiments, the present invention provides methods forscreening compounds. In some embodiments, the methods comprise providinga skin equivalent having a surface electrical capacitance of from about40 to about 240 pF; and treating the skin equivalent with said compound.In further embodiments, the methods comprise step c) assaying the effectof said compound on said skin equivalent. In some preferred embodiments,the compound is selected from a combinatorial library. The presentinvention is not limited to skin equivalents formed from a particularsource of keratinocytes. Indeed, the skin equivalents may be formed froma variety of primary and immortal keratinocytes, including, but notlimited to NIKS cells. In still further embodiments, the keratinocytesexpress exogenous wild-type or variant GKLF. In still furtherembodiments, the keratinocytes are derived from two different sources.In other embodiments, the skin equivalent has a surface electricalcapacitance of from about 40 to about 240 pF. In some preferredembodiments, the skin equivalent has a surface electrical capacitance offrom about 80 to about 120 pF. In other preferred embodiments, thecontent of ceramides 5, 6, and 7 in the skin equivalent is from about 20to about 50% of total ceramide content. In still other preferredembodiments, the content of ceramide 2 in the skin equivalent is fromabout 10 to about 40% of total ceramide content.

In other embodiments, the present invention provides kits comprising atleast one skin equivalent having a surface electrical capacitance offrom about 40 to about 240 pF. In still other embodiments, the kitincludes culture media for culturing the at least one skin equivalent.In some embodiments, the kit further comprises instructions forculturing the skin equivalent. In other embodiments, the kit furthercomprises instructions for testing compounds using said at least oneskin equivalent. The present invention is not limited to skinequivalents formed from a particular source of keratinocytes. Indeed,the skin equivalents may be formed from a variety of primary andimmortal keratinocytes, including, but not limited to NIKS cells. Instill further embodiments, the keratinocytes express wild-type orvariant GKLF. In still further embodiments, the keratinocytes arederived from two different sources. In other embodiments, the skinequivalent has a surface electrical capacitance of from about 80 toabout 120 pF. In some preferred embodiments, the skin equivalent has asurface electrical capacitance of from about 80 to about 120 pF. Inother preferred embodiments, the content of ceramides 5, 6, and 7 in theskin equivalent is from about 20 to about 50% of total ceramide content.In still other preferred embodiments, the content of ceramide 2 in theskin equivalent is from about 10 to about 40% of total ceramide content.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleic acid sequence for mouse Klf4 (SEQ ID NO:1).

FIG. 2 shows the nucleic acid sequence for GKLF (SEQ ID NO:2)

DEFINITIONS

As used herein, the term “GKLF” when used in reference to a protein ornucleic acid refers to a protein or nucleic acid encoding a protein thatshares greater than about 50% identity with SEQ ID NO:1 and/or SEQ IDNO:2 and binds to the basic transcription element of the cytochromep450IAI promoter. Binding activity may be conveniently assayed by anelectrophoretic mobility gel shift assay using the oligonucleotideGAGAAGGAGGCGTGGCCAAC (SEQ ID NO:3) as described in Zhang et al., J.Biol. Chem., 273(28): 17917-25 (1998). Thus, the term GKLF encompassesboth proteins that are identical to wild-type GKLF and those that arederived from wild type GKLF (e.g., variants of GKLF or chimeric genesconstructed with portions of GKLF coding regions).

As used herein, the term “activity of GKLF” refers to any activity ofwild type GKLF. The term is intended to encompass all activities ofGKLF, alone or in combination.

As used herein, the terms “skin equivalent” and “skin substitute” areused interchangeably to refer to an in vitro derived culture ofkeratinocytes that has stratified into squamous epithelia. Typically,the skin equivalents are produced by organotypic culture.

As used herein, the term “content of ceramides” refers to the amount ofceramides in a skin equivalent sample as assayed by high-performancethin-layer chromatography.

As used herein, the term “organotypic” culture refers to athree-dimensional tissue culture where cultured cells are usedreconstruct a tissue or organ in vitro.

As used herein, the term “NIKS cells” refers to cells havingcharacteristics of the cells deposited as cell line ATCC CRL-12191.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor (e.g., GKLF). The polypeptide can be encoded by a fulllength coding sequence or by any portion of the coding sequence so longas the desired activity or functional properties (e.g., enzymaticactivity, ligand binding, signal transduction, etc.) of the full-lengthor fragment are retained. The term also encompasses the coding region ofa structural gene and the including sequences located adjacent to thecoding region on both the 5′ and 3′ ends for a distance of about 1 kb oneither end such that the gene corresponds to the length of thefull-length mRNA. The sequences that are located 5′ of the coding regionand which are present on the mRNA are referred to as 5′ untranslatedsequences. The sequences that are located 3′ or downstream of the codingregion and that are present on the mRNA are referred to as 3′untranslated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

In particular, the term “GKLF gene” refers to the full-length GKLFnucleotide sequence (e.g., contained in SEQ ID NO:2). However, it isalso intended that the term encompass fragments of the GKLF sequence, aswell as other domains within the full-length GKLF nucleotide sequence.Furthermore, the terms “GKLF nucleotide sequence” or “GKLFpolynucleotide sequence” encompasses DNA, cDNA, and RNA (e.g., mRNA)sequences.

Where “amino acid sequence” is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, “amino acidsequence” and like terms, such as “polypeptide” or “protein” are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

In addition to containing introns, genomic forms of a gene may alsoinclude sequences located on both the 5′ and 3′ end of the sequencesthat are present on the RNA transcript. These sequences are referred toas “flanking” sequences or regions (these flanking sequences are located5′ or 3′ to the non-translated sequences present on the mRNAtranscript). The 5′ flanking region may contain regulatory sequencessuch as promoters and enhancers that control or influence thetranscription of the gene. The 3′ flanking region may contain sequencesthat direct the termination of transcription, post-transcriptionalcleavage and polyadenylation.

The term “wild-type” refers to a gene or gene product that has thecharacteristics of that gene or gene product when isolated from anaturally occurring source. A wild-type gene is that which is mostfrequently observed in a population and is thus arbitrarily designed the“normal” or “wild-type” form of the gene. In contrast, the terms“modified”, “mutant”, and “variant” refer to a gene or gene product thatdisplays modifications in sequence and or functional properties (i.e.,altered characteristics) when compared to the wild-type gene or geneproduct. It is noted that naturally-occurring mutants can be isolated;these are identified by the fact that they have altered characteristicswhen compared to the wild-type gene or gene product.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

As used herein, the terms “an oligonucleotide having a nucleotidesequence encoding a gene” and “polynucleotide having a nucleotidesequence encoding a gene,” means a nucleic acid sequence comprising thecoding region of a gene or, in other words, the nucleic acid sequencethat encodes a gene product. The coding region may be present in cDNA,genomic DNA, or RNA form. When present in a DNA form, theoligonucleotide or polynucleotide may be single-stranded (i.e., thesense strand) or double-stranded. Suitable control elements such asenhancers/promoters, splice junctions, polyadenylation signals, etc. maybe placed in close proximity to the coding region of the gene if neededto permit proper initiation of transcription and/or correct processingof the primary RNA transcript. Alternatively, the coding region utilizedin the expression vectors of the present invention may containendogenous enhancers/promoters, splice junctions, intervening sequences,polyadenylation signals, etc. or a combination of both endogenous andexogenous control elements.

As used herein, the term “regulatory element” refers to a geneticelement that controls some aspect of the expression of nucleic acidsequences. For example, a promoter is a regulatory element thatfacilitates the initiation of transcription of an operably linked codingregion. Other regulatory elements include splicing signals,polyadenylation signals, termination signals, etc.

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, for the sequence“A-G-T,” is complementary to the sequence “T-C-A.” Complementarity maybe “partial,” in which only some of the nucleic acids' bases are matchedaccording to the base pairing rules. Or, there may be “complete” or“total” complementarity between the nucleic acids. The degree ofcomplementarity between nucleic acid strands has significant effects onthe efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions, aswell as detection methods that depend upon binding between nucleicacids.

The term “homology” refers to a degree of complementarity. There may bepartial homology or complete homology (i.e., identity). A partiallycomplementary sequence is one that at least partially inhibits acompletely complementary sequence from hybridizing to a target nucleicacid and is referred to using the functional term “substantiallyhomologous.” The term “inhibition of binding,” when used in reference tonucleic acid binding, refers to inhibition of binding caused bycompetition of homologous sequences for binding to a target sequence.The inhibition of hybridization of the completely complementary sequenceto the target sequence may be examined using a hybridization assay(Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (i.e., the hybridization)of a completely homologous to a target under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target that lacks even a partial degreeof complementarity (e.g., less than about 30% identity); in the absenceof non-specific binding the probe will not hybridize to the secondnon-complementary target.

The art knows well that numerous equivalent conditions may be employedto comprise low stringency conditions; factors such as the length andnature (DNA, RNA, base composition) of the probe and nature of thetarget (DNA, RNA, base composition, present in solution or immobilized,etc.) and the concentration of the salts and other components (e.g., thepresence or absence of formamide, dextran sulfate, polyethylene glycol)are considered and the hybridization solution may be varied to generateconditions of low stringency hybridization different from, butequivalent to, the above listed conditions. In addition, the art knowsconditions that promote hybridization under conditions of highstringency (e.g., increasing the temperature of the hybridization and/orwash steps, the use of formamide in the hybridization solution, etc.).

When used in reference to a double-stranded nucleic acid sequence suchas a cDNA or genomic clone, the term “substantially homologous” refersto any probe that can hybridize to either or both strands of thedouble-stranded nucleic acid sequence under conditions of low stringencyas described below.

When used in reference to a single-stranded nucleic acid sequence, theterm “substantially homologous” refers to any probe that can hybridize(i.e., it is the complement of) the single-stranded nucleic acidsequence under conditions of low stringency as described above.

As used herein, the term “competes for binding” is used in reference toa first polypeptide with an activity which binds to the same substrateas does a second polypeptide with an activity, where the secondpolypeptide is a variant of the first polypeptide or a related ordissimilar polypeptide. The efficiency (e.g., kinetics orthermodynamics) of binding by the first polypeptide may be the same asor greater than or less than the efficiency substrate binding by thesecond polypeptide. For example, the equilibrium binding constant(K_(D)) for binding to the substrate may be different for the twopolypeptides. The term “K_(m)” as used herein refers to theMichaelis-Menton constant for an enzyme and is defined as theconcentration of the specific substrate at which a given enzyme yieldsone-half its maximum velocity in an enzyme catalyzed reaction.

As used herein, the term “hybridization” is used in reference to thepairing of complementary nucleic acids. Hybridization and the strengthof hybridization (i.e., the strength of the association between thenucleic acids) is impacted by such factors as the degree ofcomplementary between the nucleic acids, stringency of the conditionsinvolved, the T_(m) of the formed hybrid, and the G:C ratio within thenucleic acids.

As used herein, the term “T_(m)” is used in reference to the “meltingtemperature.” The melting temperature is the temperature at which apopulation of double-stranded nucleic acid molecules becomes halfdissociated into single strands. The equation for calculating the T_(m)of nucleic acids is well known in the art. As indicated by standardreferences, a simple estimate of the T_(m) value may be calculated bythe equation: T_(m)=81.5+0.41(% G+C), when a nucleic acid is in aqueoussolution at 1 M NaCl (See e.g., Anderson and Young, Quantitative FilterHybridization, in Nucleic Acid Hybridization [1985]). Other referencesinclude more sophisticated computations that take structural as well assequence characteristics into account for the calculation of T_(m).

As used herein the term “stringency” is used in reference to theconditions of temperature, ionic strength, and the presence of othercompounds such as organic solvents, under which nucleic acidhybridizations are conducted. Those skilled in the art will recognizethat “stringency” conditions may be altered by varying the parametersjust described either individually or in concert. With “high stringency”conditions, nucleic acid base pairing will occur only between nucleicacid fragments that have a high frequency of complementary basesequences (e.g., hybridization under “high stringency” conditions mayoccur between homologs with about 85-100% identity, preferably about70-100% identity). With medium stringency conditions, nucleic acid basepairing will occur between nucleic acids with an intermediate frequencyof complementary base sequences (e.g., hybridization under “mediumstringency” conditions may occur between homologs with about 50-70%identity). Thus, conditions of “weak” or “low” stringency are oftenrequired with nucleic acids that are derived from organisms that aregenetically diverse, as the frequency of complementary sequences isusually less.

“High stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 0.1×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Medium stringency conditions” when used in reference to nucleic acidhybridization comprise conditions equivalent to binding or hybridizationat 42° C. in a solution consisting of 5×SSPE (43.8 g/l NaCl, 6.9 g/lNaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS,5×Denhardt's reagent and 100 μg/ml denatured salmon sperm DNA followedby washing in a solution comprising 1.0×SSPE, 1.0% SDS at 42° C. when aprobe of about 500 nucleotides in length is employed.

“Low stringency conditions” comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄ H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.1% SDS, 5×Denhardt's reagent [50×Denhardt's contains per 500ml: 5 g Ficoll (Type 400, Pharamcia), 5 g BSA (Fraction V; Sigma)] and100 μg/ml denatured salmon sperm DNA followed by washing in a solutioncomprising 5×SSPE, 0.1% SDS at 42° C. when a probe of about 500nucleotides in length is employed.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence,” “sequenceidentity,” “percentage of sequence identity,” and “substantialidentity.” A “reference sequence” is a defined sequence used as a basisfor a sequence comparison; a reference sequence may be a subset of alarger sequence, for example, as a segment of a full-length cDNAsequence given in a sequence listing or may comprise a complete genesequence. Generally, a reference sequence is at least 20 nucleotides inlength, frequently at least 25 nucleotides in length, and often at least50 nucleotides in length. Since two polynucleotides may each (1)comprise a sequence (i.e., a portion of the complete polynucleotidesequence) that is similar between the two polynucleotides, and (2) mayfurther comprise a sequence that is divergent between the twopolynucleotides, sequence comparisons between two (or more)polynucleotides are typically performed by comparing sequences of thetwo polynucleotides over a “comparison window” to identify and comparelocal regions of sequence similarity. A “comparison window”, as usedherein, refers to a conceptual segment of at least 20 contiguousnucleotide positions wherein a polynucleotide sequence may be comparedto a reference sequence of at least 20 contiguous nucleotides andwherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman [Smithand Waterman, Adv. Appl. Math. 2: 482 (1981)] by the homology alignmentalgorithm of Needleman and Wunsch [Needleman and Wunsch, J. Mol. Biol.48:443 (1970)], by the search for similarity method of Pearson andLipman [Pearson and Lipman, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444(1988)], by computerized implementations of these algorithms (GAP,BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software PackageRelease 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.),or by inspection, and the best alignment (i.e., resulting in the highestpercentage of homology over the comparison window) generated by thevarious methods is selected. The term “sequence identity” means that twopolynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25-50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence, for example, as a segment of the full-length sequences of thecompositions claimed in the present invention (e.g., Klf-4).

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions that are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule that is comprised of segments of DNA joinedtogether by means of molecular biological techniques.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is present in a form or settingthat is different from that in which it is found in nature. In contrast,non-isolated nucleic acids are nucleic acids such as DNA and RNA foundin the state they exist in nature. For example, a given DNA sequence(e.g., a gene) is found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, are found in the cell as a mixture withnumerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding GKLF includes, by way of example, suchnucleic acid in cells ordinarily expressing GKLF where the nucleic acidis in a chromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid, oligonucleotide, or polynucleotidemay be present in single-stranded or double-stranded form. When anisolated nucleic acid, oligonucleotide or polynucleotide is to beutilized to express a protein, the oligonucleotide or polynucleotidewill contain at a minimum the sense or coding strand (i.e., theoligonucleotide or polynucleotide may single-stranded), but may containboth the sense and anti-sense strands (i.e., the oligonucleotide orpolynucleotide may be double-stranded).

As used herein the term “portion” when in reference to a nucleotidesequence (as in “a portion of a given nucleotide sequence”) refers tofragments of that sequence. The fragments may range in size from fournucleotides to the entire nucleotide sequence minus one nucleotide (10nucleotides, 20, 30, 40, 50, 100, 200, etc.).

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. The coding region is bounded, in eukaryotes, on the 5′side by the nucleotide triplet “ATG” that encodes the initiatormethionine and on the 3′ side by one of the three triplets which specifystop codons (i.e., TAA, TAG, TGA).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher than that typically observedin a given tissue in a control or non-transgenic animal. Levels of mRNAare measured using any of a number of techniques known to those skilledin the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed (e.g.,the amount of 28S rRNA, an abundant RNA transcript present atessentially the same amount in all tissues, present in each sample canbe used as a means of normalizing or standardizing the RAD50mRNA-specific signal observed on Northern blots). The amount of mRNApresent in the band corresponding in size to the correctly spliced GKLFtransgene RNA is quantified; other minor species of RNA which hybridizeto the transgene probe are not considered in the quantification of theexpression of the transgenic mRNA.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA failsto integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham and van der Eb, Virol., 52:456 [1973]),has been modified by several groups to optimize conditions forparticular types of cells. The art is well aware of these numerousmodifications.

A “composition comprising a given polynucleotide sequence” as usedherein refers broadly to any composition containing the givenpolynucleotide sequence. The composition may comprise an aqueoussolution. Compositions comprising polynucleotide sequences encoding GKLF(e.g., SEQ ID NOs: 1 and 2) or fragments thereof may be employed ashybridization probes. In this case, the GKLF encoding polynucleotidesequences are typically employed in an aqueous solution containing salts(e.g., NaCl), detergents (e.g., SDS), and other components (e.g.,Denhardt's solution, dry milk, salmon sperm DNA, etc.).

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample. Test compoundscomprise both known and potential therapeutic compounds. A test compoundcan be determined to be therapeutic by screening using the screeningmethods of the present invention. A “known therapeutic compound” refersto a therapeutic compound that has been shown (e.g., through animaltrials or prior experience with administration to humans) to beeffective in such treatment or prevention.

The term “sample” as used herein is used in its broadest sense. A samplesuspected of containing a human chromosome or sequences associated witha human chromosome may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern blot analysis), RNA (insolution or bound to a solid support such as for Northern blotanalysis), cDNA (in solution or bound to a solid support) and the like.A sample suspected of containing a protein may comprise a cell, aportion of a tissue, an extract containing one or more proteins and thelike.

As used herein, the term “response”, when used in reference to an assay,refers to the generation of a detectable signal (e.g., accumulation ofreporter protein, increase in ion concentration, accumulation of adetectable chemical product).

As used herein, the term “reporter gene” refers to a gene encoding aprotein that may be assayed. Examples of reporter genes include, but arenot limited to, luciferase (See, e.g., deWet et al., Mol. Cell. Biol.7:725 [1987] and U.S. Pat. Nos., 6,074,859; 5,976,796; 5,674,713; and5,618,682; all of which are incorporated herein by reference), greenfluorescent protein (e.g., GenBank Accession Number U43284; a number ofGFP variants are commercially available from CLONTECH Laboratories, PaloAlto, Calif.), chloramphenicol acetyltransferase, β-galactosidase,alkaline phosphatase, and horse radish peroxidase.

DESCRIPTION OF THE INVENTION

The present invention relates to in vitro cultured skin substitutes, andin particular to in vitro cultured skin substitutes that have improvedbarrier function. In some embodiments, improved barrier function is aresult of improved culture conditions, while in other embodiments,improved barrier function results from genetic modification ofkeratinocytes.

Human skin protects the body from environmental insults such aschemicals and microorganisms. It is also critical for preventing theloss of water from our bodies. Defects in skin barrier function havedetrimental effects leading to entry of poisonous substances, infectionor severe water loss. Sometimes it is desirable to improve the barrierfunction of the skin for medical, infant care or cosmetic reasons, whileat other times it would be advantageous to lower the permeabilitybarrier; to administer drugs transdermally, for example. Pharmaceutical,cosmetic and consumer product companies all have products that may comeinto contact with the skin. These companies need to know early in thedevelopment process whether their compound or formulation will affectthe essential barrier function of the skin. Excised skin tissue has beenused for measuring percutaneous absorption but a number of problems withthis preparation have been noted in the literature. There aredifferences in absorption between human and animal skin that can resultin misleading results and the availability of human tissue is variable.There are also growing political and social pressures to eliminate orreduce the number of animals being used for safety testing.

These difficulties and the growing need to understand the permeabilityproperties of new formulations and potential transdermal therapeuticshave led to many studies to improve the permeability properties of invitro skin equivalent cultures. The development of a cultured skinsubstitute that recapitulates the barrier properties of human skin willalso provide a better source of synthetic tissue for burn therapy. Theavailability of cultured skin substitutes that more closely resemblehuman skin will facilitate the testing of cosmetics, pharmaceuticals,and other topical compounds by reducing the reliance on animal testingof these products.

Stratified squamous epithelia, such as skin and oral epithelia, aremultilayered renewal tissues composed primarily of keratinocytes.Differentiated keratinocytes are continuously lost from the surface andreplaced by the proliferation of basal keratinocytes. The rate at whicha basal call initiates and completes its differentiation program appearsto be tightly regulated, although the molecular controls for suchregulation are ill-defined (Fuchs, J. Cell. Sci. Suppl., 17: 197-208(1993)). In vivo, the final stages of the terminal differentiationprocess are characterized by numerous changes includingfilaggrin-mediated keratin intermediate filament bundling, and releaseof lipids from membrane-coating granules into the intercellular space(Schurer et al., Dermatologica, 183: 77-94 (1991)). The cornifiedenvelope, another terminal differentiation structure consisting ofseveral proteins that are covalently crosslinked by the action ofcalcium-dependent transglutaminases, is also formed in differentiatingkeratinocytes (Aeschlimann et al., Thrombosis & Haemostasis, 71(4):402-15 (1994); Reichert et al., The cornified envelope: a key structureof terminally differentiating keratinocytes, in Molecular Biology of theSkin, M. Darmon, Editor. 1993, Academic Press, Inc.: San Diego.107-150(1993)). In the epidermis, keratinocytes lose intracellularorganelles and enucleate in the upper layers of the tissue, forming a“dead shell” with high tensile strength. Molecular mechanisms whichgovern keratinocyte enucleation and terminal differentiation are poorlyunderstood. Studies ((Sachsenmeier et al., J. Biol. Chem., 271: 5-8(1996); Hines et al., Promega Notes, 59: p. 30-36 (1996); Hines et al.,J. Biol. Chem., 271(11): 6245-6251 (1996); Polakowska et al.,Developmental Dynamics, 199(3): 176-88 (1994); Haake et al., J. Invest.Derm. Symp. Proc., 3: 28-35 (1998)) suggest that terminaldifferentiation in keratinocytes may constitute a special form ofapoptotic cell death.

Human skin is composed of a dermal layer containing fibroblasts embeddedin an extracellular protein matrix and an epidermal layer, consistingprimarily of keratinocytes that differentiate to form the outermost,impermeable skin layer. The primary function of human skin is to providea physical barrier to prevent excessive loss of bodily fluid due toevaporation. Barrier function is localized in the stratum corneum of theskin. The stratum corneum has been described as an array of impermeablekeratin-filled cells embedded in a matrix of lipid, analogous to a brickwall. Critical components of the stratum corneum barrier are the lipidsdeposited by the keratinocytes during formation of the stratum corneum.In the stratum granulosum, keratinocytes contain keratohyalin granulesand lamellar bodies. At the stratum granulosum/stratum corneuminterface, the lamellar bodies fuse with the plasma membrane and extrudetheir lipid contents into the intercellular space. A number of enzymesare also released which serve to process phospholipids andglucosylceramides to fatty acids and ceramides respectively. Theextracellular lipids of the stratum corneum are assembled intomultilamellar structures that surround the keratin-filled cornifiedenvelopes produced from the keratinocytes. Stratum corneum lipidscomprise 10-15% of the dry weight of the tissue and consist primarily(by weight) of ceramides (50%), cholesterol (25%) and free fatty acids(10%) in roughly equimolar amounts (Wertz et al., Chem. Phys. Lipids.,91(2): 85-96 (1998)). These lipids are derived principally frombiosynthesis in the keratinocytes. A portion of the ceramides have theunusual role of forming covalent bonds with groups at the surface of thecornified envelopes, including bonds to involucrin. This covalentlybound omega-hydroxyceramide forms a lipid monolayer surrounding theouter surface of the cornified cells. The precise role of this structureis unknown. Recently the importance of omega-hydroxyceramides on barrierfunction was demonstrated by inhibiting their formation in hairlessmouse skin with an inhibitor of the CYP4 P-450 omega hydroxylase (Behneet al., J. Invest. Dermatol., 114(1): 185-92 (2000)).

After the discovery that ruthenium tetroxide could be used to reveal thelamellae in electron microscopy, analysis of stratum corneumultrastructure has provided important insights into the quality of thestratum corneum. For example, studies have examined the presence oflamellar bodies in the stratum granulosum, the appropriate excretion oflamellar body contents at the stratum granulosum/stratum corneuminterface and the presence of alternating electron dense and electronlucent bands of the lipid lamellae. Electron microscopy also revealselectron dense desmosomes in the stratum corneum, occupying ˜15% of theintercellular spaces and possibly important in cell-cell adherence.

The present invention provides skin substitutes having improved barrierfunction, and compositions and methods for making skin substituteshaving improved barrier function. For convenience, the description ofthe invention is presented in the following sections: A) Sources ofKeratinocytes and Other Cells for Creating Skin Substitutes HavingImproved Barrier Function; B) Culture Conditions for Creating ImprovedBarrier Function in Skin Substitutes; C) Genetic Modification of Cellsfor Improved barrier Function; and D) Uses of Skin Substitutes havingImproved Barrier Function.

A. Sources of Keratinocytes and Other Cells for Creating SkinSubstitutes Having Improved Barrier Function

It is contemplated that the methods of the present invention can be usedto create skin substitutes having improved barrier function. Generally,any source of cells or cell line that can stratify into squamousepithelia are useful in the present invention. Accordingly, the presentinvention is not limited to the use of any particular source of cellsthat are capable of differentiating into squamous epithelia. Indeed, thepresent invention contemplates the use of a variety of cell lines andsources that can differentiate into squamous epithelia, including bothprimary and immortalized keratinocytes. Sources of cells includekeratinocytes and dermal fibroblasts biopsied from humans and cavadericdonors (Auger et al., In Vitro Cell. Dev. Biol.—Animal 36:96-103; U.S.Pat. Nos. 5,968,546 and 5,693,332, each of which is incorporated hereinby reference), neonatal foreskins (Asbill et al., Pharm. Research 17(9):1092-97 (2000); Meana et al., Burns 24:621-30 (1998); U.S. Pat. Nos.4,485,096; 6,039,760; and 5,536,656, each of which is incorporatedherein by reference), and immortalized keratinocytes cell lines such asNM1 cells (Baden, In Vitro Cell. Dev. Biol. 23(3):205-213 (1987)), HaCaTcells (Boucamp et al., J. cell. Boil. 106:761-771 (1988)); and NIKScells (Cell line BC-1-Ep/SL; U.S. Pat. No. 5,989,837, incorporatedherein by reference; ATCC CRL-12191). Each of these cell lines can becultured or genetically modified as described below in order to improvebarrier function of the resulting skin equivalent.

In particularly preferred embodiments, NIKS cells are utilized. Thediscovery of a novel human keratinocyte cell line (near-diploidimmortalized keratinocytes or NIKS) provides an opportunity togenetically engineer human keratinocytes for new in vitro testingmethods. A unique advantage of the NIKS cells is that they are aconsistent source of genetically-uniform, pathogen-free humankeratinocytes. For this reason, they are useful for the application ofgenetic engineering and genomic gene expression approaches to provideskin equivalent cultures with properties more similar to human skin.Such systems will provide an important alternative to the use of animalsfor testing compounds and formulations. The NIKS keratinocyte cell line,identified and characterized at the University of Wisconsin, isnontumorigenic, exhibits a stable karyotype, and exhibits normaldifferentiation both in monolayer and organotypic culture. NIKS cellsform fully stratified skin equivalents in culture. These cultures areindistinguishable by all criteria tested thus far from organotypiccultures formed from primary human keratinocytes. Unlike primary cellshowever, the immortalized NIKS cells will continue to proliferate inmonolayer culture indefinitely. This provides an opportunity togenetically manipulate the cells and isolate new clones of cells withnew useful properties (Allen-Hoffmann et al., J. Invest. Dermatol.,114(3): 444-455 (2000)).

The NIKS cells arose from the BC-1-Ep strain of human neonatal foreskinkeratinocytes isolated from an apparently normal male infant. In earlypassages, the BC-1-Ep cells exhibited no morphological or growthcharacteristics that were atypical for cultured normal humankeratinocytes. Cultivated BC-1-Ep cells exhibited stratification as wellas features of programmed cell death. To determine replicative lifespan,the BC-1-Ep cells were serially cultivated to senescence in standardkeratinocyte growth medium at a density of 3×10⁵ cells per 100-mm dishand passaged at weekly intervals (approximately a 1:25 split). Bypassage 15, most keratinocytes in the population appeared senescent asjudged by the presence of numerous abortive colonies which exhibitedlarge, flat cells. However, at passage 16, keratinocytes exhibiting asmall cell size were evident. By passage 17, only the small-sizedkeratinocytes were present in the culture and no large, senescentkeratinocytes were evident. The resulting population of smallkeratinocytes that survived this putative crisis period appearedmorphologically uniform and produced colonies of keratinocytesexhibiting typical keratinocyte characteristics including cell-celladhesion and apparent squame production. The keratinocytes that survivedsenescence were serially cultivated at a density of 3×10⁵ cells per100-mm dish. Typically the cultures reached a cell density ofapproximately 8×10⁶ cells within 7 days. This stable rate of cell growthwas maintained through at least 59 passages, demonstrating that thecells had achieved immortality. The keratinocytes that emerged from theoriginal senescencing population were originally designatedBC-1-Ep/Spontaneous Line and are now termed NIKS. The NIKS cell line hasbeen screened for the presence of proviral DNA sequences for HIV-1,HIV-2, EBV, CMV, HTLV-1, HTLV-2, HBV, HCV, B-19 parvovirus, HPV-16 andHPV-31 using either PCR or Southern analysis. None of these viruses weredetected.

Chromosomal analysis was performed on the parental BC-1-Ep cells atpassage 3 and NIKS cells at passages 31 and 54. The parental BC-1-Epcells have a normal chromosomal complement of 46, XY. At passage 31, allNIKS cells contained 47 chromosomes with an extra isochromosome of thelong arm of chromosome 8. No other gross chromosomal abnormalities ormarker chromosomes were detected. At passage 54, all cells contained theisochromosome 8.

The DNA fingerprints for the NIKS cell line and the BC-1-Epkeratinocytes are identical at all twelve loci analyzed demonstratingthat the NIKS™ cells arose from the parental BC-1-Ep population. Theodds of the NIKS cell line having the parental BC-1-Ep DNA fingerprintby random chance is 4×10⁻¹⁶. The DNA fingerprints from three differentsources of human keratinocytes, ED-1-Ep, SCC4 and SCC13y are differentfrom the BC-1-Ep pattern. This data also shows that keratinocytesisolated from other humans, ED-1-Ep, SCC4, and SCC13y, are unrelated tothe BC-1-Ep cells or each other. The NIKS DNA fingerprint data providesan unequivocal way to identify the NIKS cell line.

Loss of p53 function is associated with an enhanced proliferativepotential and increased frequency of immortality in cultured cells. Thesequence of p53 in the NIKS cells is identical to published p53sequences (GenBank accession number: M14695). In humans, p53 exists intwo predominant polymorphic forms distinguished by the amino acid atcodon 72. Both alleles of p53 in the NIKS cells are wild-type and havethe sequence CGC at codon 72, which codes for an arginine. The othercommon form of p53 has a proline at this position. The entire sequenceof p53 in the NIKS cells is identical to the BC-1-Ep progenitor cells.Rb was also found to be wild-type in NIKS cells.

Anchorage-independent growth is highly correlated to tumorigenicity invivo. For this reason, the anchorage-independent growth characteristicsof NIKS cells in agar or methylcellulose-containing medium wasinvestigated. After 4 weeks in either agar- ormethylcellulose-containing medium, NIKS cells remained as single cells.The assays were continued for a total of 8 weeks to detect slow growingvariants of the NIKS cells. None were observed.

To determine the tumorigenicity of the parental BC-1-Ep keratinocytesand the immortal NIKS keratinocyte cell line, cells were injected intothe flanks of athymic nude mice. The human squamous cell carcinoma cellline, SCC4, was used as a positive control for tumor production in theseanimals. The injection of samples was designed such that animalsreceived SCC4 cells in one flank and either the parental BC-1-Epkeratinocytes or the NIKS cells in the opposite flank. This injectionstrategy eliminated animal to animal variation in tumor production andconfirmed that the mice would support vigorous growth of tumorigeniccells. Neither the parental BC-1-Ep keratinocytes (passage 6) nor theNIKS keratinocytes (passage 35) produced tumors in athymic nude mice.

NIKS cells were analyzed for the ability to undergo differentiation inboth surface culture and organotypic culture. For cells in surfaceculture, a marker of squamous differentiation, the formation cornifiedenvelopes was monitored. In cultured human keratinocytes, early stagesof cornified envelope assembly result in the formation of an immaturestructure composed of involucrin, cystatin-α and other proteins, whichrepresent the innermost third of the mature cornified envelope. Lessthan 2% of the keratinocytes from the adherent BC-1-Ep cells or the NIKScell line produce cornified envelopes. This finding is consistent withprevious studies demonstrating that actively growing, subconfluentkeratinocytes produce less than 5% cornified envelopes. To determinewhether the NIKS cell line is capable of producing cornified envelopeswhen induced to differentiate, the cells were removed from surfaceculture and suspended for 24 hours in medium made semi-solid withmethylcellulose. Many aspects of terminal differentiation, includingdifferential expression of keratins and cornified envelope formation canbe triggered in vitro by loss of keratinocyte cell-cell andcell-substratum adhesion. The NIKS keratinocytes produced as many as andusually more cornified envelopes than the parental keratinocytes. Thesefindings demonstrate that the NIKS keratinocytes are not defective intheir ability to initiate the formation of this cell type-specificdifferentiation structure.

To confirm that the NIKS keratinocytes can undergo squamousdifferentiation, the cells were cultivated in organotypic culture.Keratinocyte cultures grown on plastic substrata and submerged in mediumreplicate but exhibit limited differentiation. Specifically, humankeratinocytes become confluent and undergo limited stratificationproducing a sheet consisting of 3 or more layers of keratinocytes. Bylight and electron microscopy there are striking differences between thearchitecture of the multilayered sheets formed in tissue culture andintact human skin. In contrast, organotypic culturing techniques allowfor keratinocyte growth and differentiation under in vivo-likeconditions. Specifically, the cells adhere to a physiological substratumconsisting of dermal fibroblasts embedded within a fibrillar collagenbase. The organotypic culture is maintained at the air-medium interface.In this way, cells in the upper sheets are air-exposed while theproliferating basal cells remain closest to the gradient of nutrientsprovided by diffusion through the collagen gel. Under these conditions,correct tissue architecture is formed. Several characteristics of anormal differentiating epidermis are evident. In both the parental cellsand the NIKS cell line a single layer of cuboidal basal cells rests atthe junction of the epidermis and the dermal equivalent. The roundedmorphology and high nuclear to cytoplasmic ratio is indicative of anactively dividing population of keratinocytes. In normal humanepidermis, as the basal cells divide they give rise to daughter cellsthat migrate upwards into the differentiating layers of the tissue. Thedaughter cells increase in size and become flattened and squamous.Eventually these cells enucleate and form cornified, keratinizedstructures. This normal differentiation process is evident in the upperlayers of both the parental cells and the NIKS cells. The appearance offlattened squamous cells is evident in the upper layers of keratinocytesand demonstrates that stratification has occurred in the organotypiccultures. In the uppermost part of the organotypic cultures theenucleated squames peel off the top of the culture. To date, nohistological differences in differentiation at the light microscopelevel between the parental keratinocytes and the NIKS keratinocyte cellline grown in organotypic culture have been observed.

To observe more detailed characteristics of the parental (passage 5) andNIKS (passage 38) organotypic cultures and to confirm the histologicalobservations, samples were analyzed using electron microscopy. Parentalcells and the immortalized human keratinocyte cell line, NIKS, wereharvested after 15 days in organotypic culture and sectionedperpendicular to the basal layer to show the extent of stratification.Both the parental cells and the NIKS cell line undergo extensivestratification in organotypic culture and form structures that arecharacteristic of normal human epidermis. Abundant desmosomes are formedin organotypic cultures of parental cells and the NIKS cell line. Theformation of a basal lamina and associated hemidesmosomes in the basalkeratinocyte layers of both the parental cells and the cell line wasalso noted. Hemidesmosomes are specialized structures that increaseadhesion of the keratinocytes to the basal lamina and help maintain theintegrity and strength of the tissue. The presence of these structureswas especially evident in areas where the parental cells or the NIKScells had attached directly to the porous support. These findings areconsistent with earlier ultrastructural findings using human foreskinkeratinocytes cultured on a fibroblast-containing porous support.Analysis at both the light and electron microscopic levels demonstratethat the NIKS cell line in organotypic culture can stratify,differentiate, and form structures such as desmosomes, basal lamina, andhemidesmosomes found in normal human epidermis.

B. Culture Conditions for Creating Improved Barrier Function in SkinSubstitutes

In some embodiments of the present invention, methods of culturing skinequivalents are provided that result in enhanced barrier function ascompared to skin equivalents cultured by conventional methods. Fullstratification and histological differentiation of normal keratinocytescan be achieved by the use of three-dimensional organotypic culturemethods (Bell et al., Proc. Nat. Acad. Sci. USA, 76: 1274-1278 (1979);Fusenig, Epithelial-mesenchymal interactions regulate keratinocytegrowth and differentiation in vitro, in The Keratinocyte Handbook, I. M.Leigh, Lane, E. B., and F. M. Watt, Editor. 1994, University Press:Cambridge (1994); Parenteau et al., Cytotechnology, 9: 163-171(1992)).Normal keratinocytes grown on the surface of collagen gels containingdermal fibroblasts can generate specialized structures, such as thebasement membrane and hemidesmosomes, which are characteristic of thenormal tissue architecture of stratified squamous epithelia. Theorganotypic culture technique for normal keratinocytes has fostered therecent development of in vitro models for cutaneouspharmacotoxicological studies. This has become an important alternativeto animal testing.

When in vitro cultures of human keratinocytes are grown at an air-liquidinterface, a highly ordered stratum corneum is formed. Althoughpermeability to water decreases with increased culturing time at theair-liquid interface (Cumpstone et al., J. Invest. Dermatol., 92(4):598-600 (1989)), permeability of in vitro skin equivalent cultures ismuch greater than that of intact human skin, i.e., the barrier functionis defective in the culture systems Ponec, Int. J. Cosmetic Sci., 14:245-264 (1992)). In an effort to improve the permeability barrier, anumber of culture variables have been examined and some have led toimproved properties of the cultures (Table 1). For example, growing thecultures at lowered relative humidity improves the barrier function ofskin equivalent cultures (Mak et al., J. Invest. Dermatol., 96(3): 323-7(1991)). It is believed that transepidermal water flux may serve as aregulatory signal for epidermal lipid synthesis and repair followingdisruption of the epidermal barrier (Grubauer et al., J. Lipid Res.,30(3): 323-33 (1989)). Hairless mice have also been used to demonstrateimproved barrier function, epidermal morphology (SC thickness, number oflamellar membrane structures, number of lamellar bodies) and lipidcontent in response to lowered humidity.

A key biochemical difference between cultured skin substitutes andintact skin is the profile of extracellular lipids that are found in theoutermost layer of normal skin. Cultures of differentiated keratinocytesare deficient in several ceramides that are major constituents of normalskin (Ponec et al., J. Invest. Dermatol., 109(3): 348-55 (1997)). Largequantities of these specialized extracellular lipids are secreted bydifferentiated keratinocytes and assembled into lipid bi-layers that areessential for normal epidermal barrier function. Comparison of the lipidcomposition between in vitro skin equivalent cultures and human skinrevealed striking differences. Human skin contains seven forms ofceramides but the cultures produced primarily ceramides 1-3 and verylittle of ceramides 6 and 7. Re-establishing a more complete lipidprofile has been the end-point of a number of studies. For example,addition of vitamin C to the media was found to be critical for acomplete spectrum of ceramide lipids in skin equivalent cultures.Ceramides 6 and 7 contain hydroxylated sphingoid base and/or fatty acid,and production of these is likely facilitated by the presence of vitaminC. In this study, the lipid profiles of the commercially available skinequivalent cultures EPIDERM, SKINETHIC and Living Skin Equivalent wereall deficient in ceramides 5, 6 and 7. Addition of vitamin C improvedthe lipid profile and the overall SC architecture as determined byelectron microscopy.

TABLE 1 Effect of Substances added to in vitro Cultures on BarrierFunction or Lipid Composition Barrier Lipid Compound FunctionComposition Reference EGF High triglycerides Ponec et al., supra.Estrogen Accelerates Williams et al., J Investig. Dermatol. Symp. Proc.,3(2): 75-9 (1998). FXAR activators Accelerates Hanley et al., supra.PPAR Activators Accelerates Hanley et al. Vitamin C Major Ponec et al.improvement Vitamin E No effect Ponec et al. Vitamin D Improved/Williams et al., Mac No effect et al., supra.

Activators of nuclear hormone receptors have been tested for theireffects on barrier function development. Addition of vitamin D has ledto improvements in some studies (Mak et al., supra) but not in others(Hanley et al., supra). Activators of the peroxisomeproliferator-activated receptor (PPAR) and the farnesoid X-activatedreceptor (FXAR) accelerate barrier maturation in fetal rat skin in vitroand in utero. Structural changes were consistent with the reduction intransepidermal water loss, including the appearance of a distinct SC, athickened stratum granulosum, and increased density of lamellarstructures.

The aberrant lipid composition of skin equivalent cultures is alsoimproved by grafting onto immunodeficient mice (Vicanova et al., J.Investig. Dermatol. Symp. Proc., 3(2): 114-20 (1998)). Cultured humankeratinocytes retain the ability of generating a differentiatedepidermis when grafted onto athymic mice. When cultured skin substituteswere examined between six months and two years after grafting,significant improvements in SC lipid composition and ultrastructure wereobserved. The high levels of triglycerides and low levels of cholesterolesters and free fatty acids observed in the in vitro cultures werenormalized by six months after grafting. Ceramides 6 and 7, undetectablein the in vitro cultures, were expressed by the human cells after sixmonths of grafting. These studies highlight the fact that current invitro culture conditions are defective in their ability to produce skinequivalents with normal barrier function. Improved culture conditionsthat more closely mimic normal developmental signals should enhancebarrier function development.

The development of barrier function in vivo is temporally regulated. Inthe rat, for example, at gestational day 19 fetal rat pups have nobarrier, but by day 21 a competent barrier has formed (Aszterbaum etal., Pediatr. Res., 31(4 Pt 1): 308-17 (1992)). Development of acompetent epidermal barrier occurs between embryonic day 15 and 16 ofmouse gestation (Hardman et al., Development, 125: 1541-1552 (1998)).The functional barrier arises coincident with a multilayered SC andmature lamellar membranes in the SC. Expression of the corneocytestructural protein loricrin, filaggrin and involucrin increase duringthis period. Expression of enzymes involved in lipid processing,beta-glucocerebrosidase and steroid sulfatase, also increase. Theprocess is also susceptible to manipulation by environmental andhormonal factors. PPAR and FXAR activators given for two days in uteroaccelerated the development of barrier function on day 19 pups asmeasured by reduction in transepidermal water loss. The treatments alsoimproved SC morphology and gene expression of key structural proteinsand enzymatic functions.

Accordingly, in some embodiments of the present invention, the followingtreatments, alone or in combination, are used to provide increasedbarrier function in organotypically cultured skin equivalents. In someembodiments, the organotypic cultures are supplemented with from about 1micrograms/ml to about 200 micrograms/ml ascorbic acid, preferably about50 micrograms/ml ascorbic acid. In other embodiments, the organotypiccultures are supplemented with about 1 to 200 μM linoleic acid,preferably about 30 μM linoleic acid. In still further embodiments, theorganotypic cultures are supplemented with about 1 to 200 μM farnesol,preferably about 50 μM farnesol. In still other embodiments, theorganotypic cultures are performed at from about 50 to 95% humidity,preferably about 75% humidity. Barrier function is convenientlyevaluated in the skin equivalents by measuring surface electricalcapacitance (SEC). In preferred embodiments, skin equivalents withimproved barrier function as compared to control skin equivalents have aSEC value of less than about 5 times of the SEC observed in normal humanskin (e.g., about 150-250 pF). In particularly preferred embodiments,skin equivalents with improved barrier function as compared to controlskin equivalents have an SEC of less than about 2-3 times of the SECobserved in normal human skin (e.g., about 80-120 pF). In otherembodiments, the skin equivalents with improved barrier function arecharacterized by ceramide content. Accordingly, in some embodiments, thecontent of ceramides 5-7 is between about 20-50% of the total ceramidemass, preferably about 30-45% of the total ceramide mass. In otherembodiments, the content of ceramide 2 is between about 10 to 40% of thetotal ceramide mass, preferably about 20 to 30% of total ceramidecontent.

C. Genetic Modification of Cells for Improved Barrier Function

The present invention also contemplates that barrier function can beimproved by expressing heterologous GKLF in the cells described inSection A. Expression of heterologous GKLF may also be combined with theimproved culture conditions described in Section B. The final stages ofepidermal differentiation are preceded by increased expression ofnumerous genes that encode the enzymes required for the biochemicalmodifications that result in the stratum corneum. In addition, cultureconditions that stimulate keratinocyte differentiation or experimentaldisruption of the skin barrier function result in increased expressionof enzymes involved in extracellular lipid synthesis and metabolism.These gene expression changes indicate that one or more regulatorytranscription factors are responsible for altering the gene expressionprofile of differentiating keratinocytes to facilitate development ofthe epidermal barrier. The precise timing of skin barrier function indevelopment suggests a precise temporal control by a developmentalswitch. Complex developmental programs can be initiated by the action ofone or a small number of key regulatory transcription factors, sometimescalled master regulators or selector genes. A recent study on aknock-out mutation in the transcription factor Kruppel-like factor 4(Klf4) may have identified one of the key regulators of barrier functionin the skin (Segre et al., Nat. Genet., 22(4): 356-60 (1999)).

Klf4 mutant mice are born in normal numbers but die shortly (<15 hrs)after birth apparently from hypo-volemic shock as a result of excessiveevaporative fluid loss. Further analysis demonstrated that, while normalmice develop an intact epidermal barrier function by day 17.5 ofgestation, Klf4 mutant mice fail to develop this barrier function andhave an epidermis that exhibits excessive trans-epidermal water loss.Klf4 is expressed in the differentiating layers of the epidermis, theupper spinous and granular layers. In contrast to the defects caused inother mutant mouse lines, the absence of Klf4 does not result in grossalterations of the epidermal ultrastructure or in lipid profiles. Thisled to the hypothesis that its primary role is in the acquisition ofbarrier function. Consistent with this, defects were observed in thestratum granulosum in the keratohyalin granules and flattening of SCcells. By EM, the intercellular lamellae were discontinuous in themutant skin. The defect in Klf-4 mutant skin was not rescued by graftingit onto foster mice. Klf4 is a member of a family of transcriptionfactors; other members are implicated in tissue-specific differentiationevents in erythroid cells and T-cells. Analysis of gene expressiondifferences between wild-type and Klf4 mutant mice led to theidentification of three genes that were up-regulated in the mutant skin,suggesting that Klf4 may repress the expression of these genes normally.The observation that loss of Klf4 has no other observable effects onmouse development suggests that Klf4 functions primarily to regulate thedevelopment of the epidermal permeability barrier. The role of Klf4 inthe acquisition of epidermal barrier function raises the possibilitythat expression of Klf4 in cultured skin substitutes might improve thebarrier properties of these synthetic skin cultures.

Accordingly, in some embodiments, primary keratinocytes or immortalizedkeratinocytes are transfected with a vector encoding a functional Klf4homolog. It is contemplated that when these keratinocytes areorganotypically cultured, the resulting skin equivalent will exhibitimproved barrier function as compared to organotypic cultures formedfrom nontransfected, control keratinocytes. In preferred embodiments,skin equivalents with improved barrier function as compared to controlskin equivalents have a SEC value of less than about 5 times of the SECobserved in normal human skin (e.g., about 150-250 pF). In particularlypreferred embodiments, skin equivalents with improved barrier functionas compared to control skin equivalents have an SEC of less than about2-3 times of the SEC observed in normal human skin (e.g., about 80-120pF). In other embodiments, the skin equivalents with improved barrierfunction are characterized by ceramide content. Accordingly, in someembodiments, the content of ceramides 5-7 is between about 20-50% of thetotal ceramide mass, preferably about 30-45% of the total ceramide mass.In other embodiments, the content of ceramide 2 is between about 10 to40% of the total ceramide mass, preferably about 20 to 30% of totalceramide content.

The present invention is not limited to the use of any particularhomolog or variant of GKLF. Indeed, a variety of GKLF variants may beused so long as they retain at least some of the activity of wild-typeGKLF. In particular, it contemplated that both mouse (SEQ ID NO:1) andhuman (SEQ ID NO:2) GKLF find use in the present invention.Additionally, it is contemplated that GKLF variants encoded by sequencesthat hybridize to SEQ ID NOs: 1 and 2 under conditions of from low tohigh stringency will find use in the present invention. Functionalvariants can be screened for by expressing the variant in an appropriatevector (described in more detail below) in keratinocytes, using theheratinocytes to produce a skin equivalent, and analyzing the skinequivalent for increased barrier function. Alternatively, functionalvariants can be identified by an electrophoretic mobility shift assay asdescribed in (Zhang et al., J. Biol. Chem., 273(28): 17917-25 (1998).

In some embodiments, variants result from mutation, (i.e., a change inthe nucleic acid sequence) and generally produce altered mRNAs orpolypeptides whose structure or function may or may not be altered. Anygiven gene may have none, one, or many variant forms. Common mutationalchanges that give rise to variants are generally ascribed to deletions,additions or substitutions of nucleic acids. Each of these types ofchanges may occur alone, or in combination with the others, and at therate of one or more times in a given sequence.

It is contemplated that it is possible to modify the structure of apeptide having a function (e.g., GKLF function) for such purposes asincreasing binding affinity of GKLF for its nucleic acid ligand. Suchmodified peptides are considered functional equivalents of peptideshaving an activity of GKLF as defined herein. A modified peptide can beproduced in which the nucleotide sequence encoding the polypeptide hasbeen altered, such as by substitution, deletion, or addition. Inparticularly preferred embodiments, these modifications do notsignificantly reduce the activity of the modified GKLF. In other words,construct “X” can be evaluated in order to determine whether it is amember of the genus of modified or variant GKLF's of the presentinvention as defined functionally, rather than structurally. Inpreferred embodiments, the activity of variant or mutant GKLF isevaluated by the methods described above.

Moreover, as described above, variant forms of GKLF are alsocontemplated as being equivalent to those peptides and DNA moleculesthat are set forth in more detail herein. For example, it iscontemplated that isolated replacement of a leucine with an isoleucineor valine, an aspartate with a glutamate, a threonine with a serine, ora similar replacement of an amino acid with a structurally related aminoacid (i.e., conservative mutations) will not have a major effect on thebiological activity of the resulting molecule. Accordingly, someembodiments of the present invention provide variants of Klf4 disclosedherein containing conservative replacements. Conservative replacementsare those that take place within a family of amino acids that arerelated in their side chains. Genetically encoded amino acids can bedivided into four families: (1) acidic (aspartate, glutamate); (2) basic(lysine, arginine, histidine); (3) nonpolar (alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); and (4)uncharged polar (glycine, asparagine, glutamine, cysteine, serine,threonine, tyrosine). Phenylalanine, tryptophan, and tyrosine aresometimes classified jointly as aromatic amino acids. In similarfashion, the amino acid repertoire can be grouped as (1) acidic(aspartate, glutamate); (2) basic (lysine, arginine, histidine), (3)aliphatic (glycine, alanine, valine, leucine, isoleucine, serine,threonine), with serine and threonine optionally be grouped separatelyas aliphatic-hydroxyl; (4) aromatic (phenylalanine, tyrosine,tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17-21, 2nd ed, WH Freeman and Co., 1981). Whether achange in the amino acid sequence of a peptide results in a functionalhomolog can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner.

More rarely, a variant includes “nonconservative” changes (e.g.,replacement of a glycine with a tryptophan). Analogous minor variationscan also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues can be substituted, inserted,or deleted without abolishing biological activity can be found usingcomputer programs (e.g., LASERGENE software, DNASTAR Inc., Madison,Wis.).

The heterologous GKLF is expressed in keratinocytes by using a suitablevector and regulatory sequences. In some preferred embodiments, either ainvolucrin or transglutaminase 3 promoter are utilized. In otherpreferred embodiments, the expression of GKLF will be driven by theinducible promoter system of the pTetOn plasmid (Clontech, Palo Alto,Calif.). It is contemplated that a number of other mammalian expressionvectors are suitable for use in the present invention, including, butnot limited to, pWLNEO, pSV2CAT, pOG44, PXT1, pSG (Stratagene) pSVK3,pBPV, pMSG, pSVL (Pharmacia). Any other plasmid or vector may be used aslong as they are replicable and viable in the host. In some preferredembodiments of the present invention, mammalian expression vectorscomprise an origin of replication, a suitable promoter and enhancer, andalso any necessary ribosome binding sites, polyadenylation sites, splicedonor and acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In other embodiments, DNA sequencesderived from the SV40 splice, and polyadenylation sites may be used toprovide the required non-transcribed genetic elements. Additionally, theGKLF gene may be inserted via a retroviral vector. In particularlypreferred embodiments, the retroviral vector is pseudotyped retroviralvector (Clontech, Palo Alto, Calif.). Transfection can be accomplishedby any method known in the art, including but not limited tocalcium-phosphate coprecipitation, electroporation, microparticlebombardment, liposome mediated transfection, or retroviral infection.

D. Uses of Skin Substitutes having Improved Barrier Function

It is contemplated that the skin substitutes of the present inventionhave a variety of uses. These uses include, but are not limited to, usefor screening compounds, substrates for culturing tumors andpathological agents (e.g., human papilloma virus), and use for woundclosure and burn treatment. These uses are described in more detailbelow.

1. Use for Screening Compounds

The skin equivalents of the present invention may be used for a varietyof in vitro tests. In particular, the skin equivalents find use in theevaluation of: skin care products, drug metabolism, cellular responsesto test compounds, wound healing, phototoxicity, dermal irritation,dermal inflammation, skin corrosivity, and cell damage. The skinequivalents are provided in a variety of formats for testing, includingbut not limited to, 6-well, 24-well, and 96-well plates. Additionally,the skin equivalents can be divided by standard dissection techniquesand then tested. The skin equivalents of the present invention have bothan epidermal layer with a differentiated stratum corneum and dermallayer that includes dermal fibroblasts. As described above, inparticularly preferred embodiments, the epidermal layer is derived fromimmortalized NIKS cells. Other preferred cell lines, including NIKScells, are characterized by i) being immortalized; ii) beingnontumorigenic; iii) forming cornified envelopes when induced todifferentiate; iv) undergoing normal squamous differentiation inorganotypic culture; and v) maintaining cell type-specific growthrequirements in submerged culture, wherein said cell type-specificgrowth requirements include 1) exhibition of morphologicalcharacteristics of normal human keratinocytes when cultured in standardkeratinocyte growth medium in the presence of mitomycin C-treated 3T3feeder cells; 2) dependence on epidermal growth factor for serialcultivation; and 3) inhibition of growth by transforming growth factorβ1.

The present invention encompasses a variety of screening assays. In someembodiments, the screening method comprises providing a skin equivalentof the present invention and at least one test compound or product(e.g., a skin care product such as a moisturizer, cosmetic, dye, orfragrance; the products can be in any from, including, but not limitedto, creams, lotions, liquids and sprays), applying the product or testcompound to skin equivalent, and assaying the effect of the product ortest compound on the skin equivalent. A wide variety of assays are usedto determine the effect of the product or test compound on the skinequivalent. These assays include, but are not limited to, MTTcytotoxicity assays (Gay, The Living Skin Equivalent as an In VitroModel for Ranking the Toxic Potential of Dermal Irritants, Toxic. InVitro (1992)) and ELISA to assay the release of inflammatory modulators(e.g., prostaglandin E2, prostacyclin, and interleukin-1-alpha) andchemoattractants. The assays can be further directed to the toxicity,potency, or efficacy of the compound or product. Additionally, theeffect of the compound or product on growth, barrier function, or tissuestrength can be tested.

In particular, the present invention contemplates the use of the skinequivalents for high throughput screening of compounds fromcombinatorial libraries (e.g., libraries containing greater than 10⁴compounds). In some embodiments, the cells are used in second messengerassays that monitor signal transduction following activation ofcell-surface receptors. In other embodiments, the cells can be used inreporter gene assays that monitor cellular responses at thetranscription/translation level. In still further embodiments, the cellscan be used in cell proliferation assays to monitor the overallgrowth/no growth response of cells to external stimuli.

In second messenger assays, the skin equivalents are treated with acompound or plurality of compounds (e.g., from a combinatorial library)and assayed for the presence or absence of a second messenger response.In some preferred embodiments, the cells (e.g., NIKS cells) used tocreate the skin equivalents are transfected with an expression vectorencoding a recombinant cell surface receptor, ion-channel, voltage gatedchannel or some other protein of interest involved in a signalingcascade. It is contemplated that at least some of the compounds in thecombinatorial library can serve as agonists, antagonists, activators, orinhibitors of the protein or proteins encoded by the vectors. It is alsocontemplated that at least some of the compounds in the combinatoriallibrary can serve as agonists, antagonists, activators, or inhibitors ofprotein acting upstream or downstream of the protein encoded by thevector in a signal transduction pathway.

In some embodiments, the second messenger assays measure fluorescentsignals from reporter molecules that respond to intracellular changes(e.g., Ca²⁺ concentration, membrane potential, pH, IP3, cAMP,arachidonic acid release) due to stimulation of membrane receptors andion channels (e.g., ligand gated ion channels) (Denyer et al., DrugDiscov. Today 3:323-32 (1998); Gonzales et al., Drug. Discov. Today4:431-39 (1999)). Examples of reporter molecules include, but are notlimited to, florescence resonance energy transfer systems (e.g.,Cuo-lipids and oxonols, EDAN/DABCYL), calcium sensitive indicators(e.g., Fluo-3, FURA 2, INDO 1, and FLUO3/AM, BAPTA AM),chloride-sensitive indicators (e.g., SPQ, SPA), potassium-sensitiveindicators (e.g., PBFI), sodium-sensitive indicators (e.g., SBFI), andpH sensitive indicators (e.g., BCECF).

In general, the cells comprising the skin equivalents are loaded withthe indicator prior to exposure to the compound. Responses of the hostcells to treatment with the compounds can be detected by methods knownin the art, including, but not limited to, fluorescence microscopy,confocal microscopy, flow cytometry, microfluidic devices, FLIPR systems(Schroeder and Neagle, J. Biomol. Screening 1:75-80 (1996)), andplate-reading systems. In some preferred embodiments, the response(e.g., increase in fluorescent intensity) caused by compound of unknownactivity is compared to the response generated by a known agonist andexpressed as a percentage of the maximal response of the known agonist.The maximum response caused by a known agonist is defined as a 100%response. Likewise, the maximal response recorded after addition of anagonist to a sample containing a known or test antagonist is detectablylower than the 100% response.

The skin equivalents of the present invention are also useful inreporter gene assays. Reporter gene assays involve the use of host cellstransfected with vectors encoding a nucleic acid comprisingtranscriptional control elements of a target gene (i.e., a gene thatcontrols the biological expression and function of a disease target orinflammatory response) spliced to a coding sequence for a reporter gene.Therefore, activation of the target gene results in activation of thereporter gene product. This serves as indicator of response such aninflammatory response. Therefore, in some embodiments, the reporter geneconstruct comprises the 5′ regulatory region (e.g., promoters and/orenhancers) of a gene that is induced due to skin inflammation orirritation or protein that is involved in the synthesis of compoundsproduced in response to inflammation or irritation (e.g., prostaglandinor prostacyclin) operably linked to a reporter gene. Examples ofreporter genes finding use in the present invention include, but are notlimited to, chloramphenicol transferase, alkaline phosphatase, fireflyand bacterial luciferases, β-galactosidase, β-lactamase, and greenfluorescent protein. The production of these proteins, with theexception of green, red, yellow, or blue fluorescent protein, isdetected through the use of chemiluminescent, colorimetric, orbioluminecent products of specific substrates (e.g., X-gal andluciferin). Comparisons between compounds of known and unknownactivities may be conducted as described above.

In other preferred embodiments, the skin equivalents find use forscreening the efficacy of drug introduction across the skin or theaffect of drugs directed to the skin. In these embodiments, the skinequivalents are treated with the drug delivery system or drug, and thepermeation, penetration, or retention or the drug into the skinequivalent is assayed. Methods for assaying drug permeation are providedin Asbill et al., Pharm Res. 17(9): 1092-97 (2000). In some embodiments,the skin equivalents are mounted on top of modified Franz diffusioncells. The skin equivalents are allowed to hydrate for one hour and thenpretreated for one hour with propylene glycol. A saturated suspension ofthe model drug in propylene glycol is then added to the skin equivalent.The skin equivalent can then be sampled at predetermined intervals. Theskin equivalents are then analyzed by HPLC to determine theconcentration of the drug in the sample. Log P values for the drugs canbe determined using the ACD program (Advanced Chemistry Inc., Ontario,Canada). These methods may be adapted to study the delivery of drugs viatransdermal patches or other delivery modes.

In still further preferred embodiments, the seeded dermal equivalents,which have not yet undergone differentiation, find use in assays forcompounds that inhibit, accelerate, or otherwise effect differentiationof the seeded keratinocytes.

2. Substrates for Culturing Tumors and Pathological Agents

It is contemplated that skin equivalents of the present invention arealso useful for the culture and study of tumors that occur naturally inthe skin as well as for the culture and study of pathogens that affectthe skin. Accordingly, in some embodiments, it is contemplated that theskin equivalents of the present invention are seeded with malignantcells. By way of non-limiting example, the skin equivalents can beseeded with malignant SCC13y cells as described in U.S. Pat. No.5,989,837, which is incorporated herein by reference, to provide a modelof human squamous cell carcinoma. These seeded skin equivalents can thenbe used to screen compounds or other treatment strategies (e.g.,radiation or tomotherapy) for efficacy against the tumor in its naturalenvironment. Thus, some embodiments of the present invention providemethods comprising providing a skin equivalent comprising malignantcells or a tumor and at least one test compound, treating the skinequivalent with the compound, and assaying the effect of the treatmenton the malignant cells or tumors. In other embodiments of the presentinvention, methods are provided that comprise providing a skinequivalent comprising malignant cells or a tumor and at least one testtherapy (e.g., radiation or phototherapy) treating the skin equivalentwith the therapy, and assaying the effect of the therapy on themalignant cells or tumors.

In other embodiments, the skin equivalents are used to culture and studyskin pathogens. By way of non-limiting example, the skin equivalents areinfected with human papilloma virus (HPV) such as HPV18. Methods forpreparing skin equivalents infected with HPV are described in U.S. Pat.No. 5,994,115, which is incorporated herein by reference. Thus, someembodiments of the present invention provide methods comprisingproviding a skin equivalent infected with a pathogen of interest and atleast one test compound or treatment and treating the skin equivalentwith the test compound or treatment. In some preferred embodiments, themethods further comprise assaying the effect the test compound ortreatment on the pathogen. Such assays may be conducted by assaying thepresence, absence, or quantity of the pathogen in the skin substitutefollowing treatment. For example, an ELISA may be performed to detect orquantify the pathogen. In some particularly preferred embodiments, thepathogen is viral pathogen such as HPV.

3. Wound Closure and Burn Treatment

The skin equivalents of the present invention find use in wound closureand burn treatment applications. The use of autografts and allograftsfor the treatment of burns and wound closure is described in Myers etal., A. J. Surg. 170(1):75-83 (1995) and U.S. Pat. Nos. 5,693,332;5,658,331; and 6,039,760, each of which is incorporated herein byreference. In some embodiments, the skin equivalents may be used inconjunction with dermal replacements such as DERMAGRAFT. In otherembodiments, the skin equivalents are produced using both a standardsource of keratinocytes (e.g., NIKS cells) and keratinocytes from thepatient that will receive the graft. Therefore, the skin equivalentcontains keratinocytes from two different sources. In still furtherembodiments, the skin equivalent contains keratinocytes from a humantissue isolate. Accordingly, the present invention provides methods forwound closure, including wounds caused by burns, comprising providing askin equivalent having improved barrier function according to thepresent invention and a patient suffering from a wound and treating thepatient with the skin equivalent under conditions such that the wound isclosed.

4. Gene Therapy

In still further embodiments, the skin equivalent is engineered toprovide a therapeutic agent to a subject. The present invention is notlimited to the delivery of any particular therapeutic agent. Indeed, itis contemplated that a variety of therapeutic agents may be delivered tothe subject, including, but not limited to, enzymes, peptides, peptidehormones, other proteins, ribosomal RNA, ribozymes, and antisense RNA.These therapeutic agents may be delivered for a variety of purposes,including but not limited to the purpose of correcting genetic defects.In some particular preferred embodiments, the therapeutic agent isdelivered for the purpose of detoxifying a patient with an inheritedinborn error of metabolism (e.g., aninoacidopathesis) in which the graftserves as wild-type tissue. It is contemplated that delivery of thetherapeutic agent corrects the defect. In some embodiments, thekeratinocytes used to form the skin equivalent are transfected with aDNA construct encoding a therapeutic agent (e.g., insulin, clottingfactor IX, erythropoietin, etc) and the skin equivalent is grafted ontothe subject. The therapeutic agent is then delivered to the patient'sbloodstream or other tissues from the graft. In preferred embodiments,the nucleic acid encoding the therapeutic agent is operably linked to asuitable promoter. The present invention is not limited to the use ofany particular promoter. Indeed, the use of a variety of promoters iscontemplated, including, but not limited to, inducible, constitutive,tissue specific, and keratinocyte specific promoters. In someembodiments, the nucleic acid encoding the therapeutic agent isintroduced directly into the keratinocytes (i.e., by calcium phosphateco-precipitation or via liposome transfection). In other preferredembodiments, the nucleic acid encoding the therapeutic agent is providedas a vector and the vector is introduced into the keratinocytes bymethods known in the art. In some embodiments, the vector is an episomalvector such as a plasmid. In other embodiments, the vector integratesinto the genome of the keratinocytes. Examples of integrating vectorsinclude, but are not limited to, retroviral vectors, adeno-associatedvirus vectors, and transposon vectors.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); C.(degrees Centigrade); U (units), mU (milliunits); min. (minutes); sec.(seconds); % (percent); kb (kilobase); bp (base pair); PCR (polymerasechain reaction); BSA (bovine serum albumin).

Example 1 Effect of Culture Conditions on Epidermal Barrier Function ofNIKS Cells

While a number of culture conditions have been shown to enhance variousaspects of barrier function (see Table 1, supra), there has been nosystematic attempt to examine synergistic effects of these conditions.This example provides an assessment of the combined effects of ascorbicacid, PPAR activators (linoleic acid), FXAR activators (farnesol), andreduced relative humidity on epidermal barrier function of NIKSorganotypic cultures. Barrier properties of treated cultures areevaluated by measuring surface electrical capacitance (SEC) (Boyce etal., J. Invest. Dermatol., 107(1): p. 82-7 (1996)), analysis ofextracellular lipid composition, and by ultrastructural examination oftissue sections by electron microscopy.

The culture conditions to be evaluated are presented in Table 2. Culturesupplements are added individually or in combination to thecornification medium at the indicated concentrations. The organotypiccultures are incubated at the air/liquid interface for 14-17 days beforeanalysis. The NIKS-based cultures consist of dermal and epidermalcompartments. The dermal compartment consists of a collagen base and isformed by mixing normal human neonatal fibroblasts, strain CI-1-F, withType I collagen in Ham's F-12 medium containing 10% fetal calf serum(FCS) and penicillin/streptomycin (P/S) and allowing contraction. Theepidermal compartment is produced by seeding the NIKS cells on thecontracted collagen gel in 25 μl of a mixture of Ham's F-12:DME, (3:1,final calcium concentration 1.88 mM) supplemented with 0.2% FCS, 0.4μg/ml hydrocortisone (HC), 8.4 ng/ml cholera toxin (CT), 5 μg/ml insulin(Ins), 24 μg/ml adenine (Ade), and 100 units/ml P/S. Cells are allowedto attach 2 hours before flooding culture chamber with media (day 0). Ondays 1 and 2 cells are refed. On day 4, cells are lifted to the airinterface with a cotton pad and switched to cornification mediumcontaining Ham's F-12:DME, (3:1, final calcium concentration 1.88 mM)supplemented with 2% FCS, 0.4 μg/ml HC, 8.4 ng/ml CT, 5 μg/ml Ins, 24μg/ml Ade, and P/S. Cells are fed fresh cornification medium every 3days.

For measurement of SEC, the impedence of the culture surfaces isdetermined using a Dermaphase 9003 impedence meter (NOVA TechnologiesCorp, Portsmouth, N.H.). This instrument provides a measure of theelectrical conductivity of the skin surface, which is directly relatedto the sample's hydration state and barrier properties. The probe isplaced in contact with the culture surface and readings are takenimmediately upon probe contact and at the end of the 10 second period.The initial reading is then compared to the reading after the probe hasbeen in place for 10 seconds. An increase in the reading after 10seconds reflects increased hydration of the culture surface due toocclusion of the skin surface by the probe. Since surface hydration islargely determined by the permeability of the stratum corneum, themagnitude of the difference between the initial and final SEC readingsprovides a measure of the barrier properties of the cultures. Eachculture condition is analyzed in triplicate and the average impedencemeasurements compared to standard, unsupplemented culture conditions toassess improvements in barrier function. SEC readings from in vitrocultures are also compared to SEC measurements obtained from normalhuman skin. Previous studies have shown that the SEC values of in vitroskin substitutes (400 pF) are about ten-fold higher than those observedwith normal human skin. The goal of these experiments is to developconditions that improve barrier function of organotypic keratinocytecultures such that SEC readings of these cultures are at most 2-3 foldhigher than normal skin.

TABLE 2 Culture Supplements and Humidity Ascorbic acid Linoleic AcidFarnesol Humidity 100%  75% 50 micrograms/ml 100% 50 micrograms/ml  75%30 micromolar 100% 30 micromolar  75% 50 micromolar 100 50 micromolar 75% 50 micrograms/ml 30 micromolar 100% 50 micrograms/ml 30 micromolar 75% 50 micrograms/ml 50 micromolar 100% 50 micrograms/ml 50 micromolar 75% 30 micromolar 50 micromolar 100% 30 micromolar 50 micromolar  75%50 micrograms/ml 30 micromolar 50 micromolar 100% 50 micrograms/ml 30micromolar 50 micromolar  75%

The lipid compositions of in vitro skin substitutes examined to dateshow significant differences compared to that found in normal humanskin. In particular, the levels of ceramides 6 and 7 are greatly reducedin the in vitro cultures. To determine whether preparation oforganotypic skin cultures using the culture conditions shown in Table 2has a synergistic effect on epidermal lipid composition, the lipidprofiles of supplemented cultures are compared to lipids isolated fromunsupplemented control cultures and to lipids extracted from normalhuman skin. Lipid profiles of the cultures are determined byhigh-performance thin-layer chromatography (HPTLC) of lipids extractedfrom the epidermal culture layers. Organotypic cultures are heated to60° C. for 1 min to separate the epidermal and dermal layers. Totallipids are extracted from the epidermal layer by sequential extractionwith 2 ml chloroform/methanol (1:2), 2 ml chloroform/methanol/water(1:2:0.5), 2 ml chloroform/methanol (1:2), 2 ml chloroform/methanol(2:1) and finally with 2 ml chloroform. Following the addition of 0.2 ml2.5% KCl and 2 ml water, the samples are centrifuged and the lower phaseremoved to a clean tube. The remaining upper phase is extracted with 4ml chloroform. The chloroform extract is combined with the lower phasefrom the initial extract. Solvents are removed by evaporation undernitrogen and the extracted lipids are dissolved in chloroform/methanol(2:1). Total lipid content in the extracts is determined by weighingsamples of the extract after evaporation of solvent.

Extracted lipids (50 micrograms) are applied to silica gel 60 HPTLCplates (Merck, Darmstad, FRG) and resolved by one-dimensional HPTLCusing the ceramide development system. Lipid separation is achieved bysequential development of HPTLC plates in chloroform,chloroform/acetone/methanol (76:8:16), chloroform/hexylacetate/acetone/methanol (86:1:10:4), chloroform/acetone/methanol(76:4:20), chloroform/diethyl ether/hexyl acetate/ethylacetate/acetone/methanol (72:4:1:4:16:4), and finally withhexane/diethyl ether/ethyl acetate (80:16:4). The TLC plate is driedbriefly following each development step before proceeding to the nextsolvent system. Following separation, lipids are detected by stainingwith copper acetate and copper sulfate in sulfuric acid followed bycharring. Each culture condition is analyzed in triplicate and thelevels of specific lipid components quantified by densitometry andexpressed as a percentage of total lipid. Previous studies have shownthat ceramides 5-7 comprise only 10% of total ceramide mass of in vitroskin substitutes as compared to 39% in normal epidermis. There is acorresponding increase in ceramide 2 in the in vitro cultures, whichcomprises approximately 50% of total ceramide mass as compared to 22% innormal skin. In preferred embodiments, skin equivalents of the presentinvention are cultured under conditions that result in an increase inthe content of ceramides 5-7 to between 30-45% of total ceramide massand a reduction of the levels of ceramide 2 to between 20-30% of totalceramide mass.

The ultrastructure of lipid lamellae in organotypic cultures preparedunder the conditions described in Table 2 is examined by electronmicroscopy. Cultures are fixed in 2% glutaraldehyde and 2% formaldehydein 0.1M cacodylate buffer pH 7.4, then post-fixed in 1% osmium tetroxidefollowed by 0.25% ruthenium tetroxide. Samples are dehydrated through anethanol series, embedded in Eponate, and sectioned on a ReichertUltracut microtome. Sections are stained with uranyl acetate and leadcitrate and examined using a Hitachi H-7000 electron microscope(Hitachi, San Jose, Calif.). The organotypic cultures produced by theconditions listed in Table 2 are examined for restoration of the patternof alternating electron lucent and electron dense lipid lamellae seen innormal skin.

Organotypic cultures are also assessed for cell proliferation andcell-type specific differentiation markers, including involucrin,transglutaminase and keratins.

Example 2 Expression of Exogenous Klf4 in NIKS Cells

This Example describes the expression of exogenous Klf4 in NIKS cells.The transcription factor Krüppel-like factor 4 (Klf4) is a zinc-fingerprotein expressed at high levels in epithelium undergoing terminaldifferentiation, especially skin and intestinal epithelium. In skin, itis enriched in the mitotically inactive suprabasal layer of theepidermis. Klf4 was identified by low-stringency hybridization with aprobe for a zinc-finger domain in a NIH 3T3 cell cDNA library (Shieldset al., J. Biol. Chem, 271(33): 20009-17 (1996)). Its three C2H2 zincfingers relate it to a family of zinc finger transcription factors thatincludes EKLF and LKLF, factors that are important for tissue-specificdifferentiation. It is expressed at highest levels in growth-arrestedcells and at undetectable levels in proliferating cells. Constitutiveexpression of Klf4 COS-1 cells inhibits DNA synthesis. It binds to adefined DNA sequence that is important in the regulation of thecytochrome P450 gene CYP1A1 (Zhang et al., J. Biol. Chem., 273(28):17917-25 (1998)). Binding of Klf4 to its binding site in CYP1A1 inhibitsexpression of CYP1A1, probably by competing for DNA binding with SP1 andthrough direct protein-protein interactions with SP1. Recent studiesreport that Klf4 can also regulate its own expression and that animportant binding interaction is with p300/CBP (Geiman et al., NucleicAcids Res., 28(5): 1106-1113 (2000); Mahatan et al., Nucleic Acids Res.,27(23): 4562-9 (1999)). As is true with other key transcription factors,Klf4 can be a potent activator of some genes and a repressor of others.

Klf4 is currently the best candidate gene for a key regulator of barrierfunction in the skin. Elimination of Klf4 expression in mice results inneonatal lethality, apparently as a result of excessive water lossthrough a defective epidermal permeability barrier. These observationssuggest that Klf4 regulates genes that are essential for the formationof a normal epidermal permeability barrier and raise the possibilitythat expression of Klf4 in cultured skin substitutes might improve thebarrier function of these cultures. This Example describes two methodsof expressing Klf4 in differentiating keratinocytes. The first method isthe generation of an inducible expression construct in which expressionof human Klf4 is regulated by the presence or absence of thetetracycline derivative, doxycycline, in the culture medium. A secondmethod of directing Klf4 expression in organotypic cultures utilizes aDNA fragment containing either 3.7 kb of the involucrin promoter region,which directs expression in differentiating keratinocytes (Carroll etal., Proc. Natl. Acad. Sci. USA, 90(21): p. 10270-4 (1993)) or 135 bp ofthe transglutaminase 3 promoter region, which also directs expression indifferentiating keratinocytes (Lee et al., J. Biol. Chem., 271(8):4561-8 (1996)).

The cDNA encoding human Klf4 is isolated by PCR using primers to theknown Klf4 sequence (Yet et al., J. Biol. Chem., 273(2): 1026-31(1998)). The Klf4 cDNA is cloned into the expression vector pTRE2(Clontech, Palo Alto, Calif.), which contains a minimal CMV promoterflanked by seven repeats of the tet operator (tetO). The integrity ofthe cloned Klf4 cDNA is verified by sequence analysis using primersderived from the known Klf4 sequence.

Purified DNA from the Klf4 expression plasmid is introduced into NIKScells along with the pTet-On plasmid (Clontech, Palo Alto, Calif.),which encodes a derivative of the tet repressor protein. This protein,rtTA, binds to the tet operator in the presence of doxycycline andinduces expression of Klf4 when doxycycline is present in the culturemedium. The gene encoding a protein that confers resistance toblasticidin will be amplified by PCR and cloned into the pTet-On plasmidto allow for selection of stably-transfected cells. Transfected cellsare selected by growth in media containing blasticidin (5micrograms/ml), which will kill any NIKS cells that have notincorporated the plasmids into their genome. Stable cell lines thatcontain both the pTRE2-Klf4 and pTet-On plasmids are identified byexamining multiple clonal cell lines by Southern blot usingdigoxygenin-labeled probes derived from both the pTet-On and pTRE2plasmids. Multiple clones that contain intact copies of the pTet-On andpTRE2 plasmids are isolated and examined for expression of the Klf4transgene in the presence of doxycycline.

To examine expression from the Klf4 transgene, monolayer cultures ofstably-transfected cell lines and control untransfected cells areincubated in medium containing doxycycline (1 microgram per ml). TotalRNA is then isolated from cultures at multiple time points afterdoxycycline addition using Trizol Reagent (Life Technologies, Rockville,Md.). Twenty micrograms of total RNA is analyzed by Northern blothybridization using digoxygenin-labeled probes derived from the clonedKlf4 cDNA and detected using the Genius non-radioactive detection system(Roche Molecular Biochemicals, Indianapolis, Ind.). RNA is also isolatedfrom transfected cultures grown in the absence of doxycycline todetermine the basal level of Klf4 expression from the transgene. RNAisolated from untransfected cells at each time point will be analyzed toestablish a background level of Klf4 expression from the endogenous Klf4gene.

The Klf4 cDNA is also cloned into an expression plasmid containingpromoter sequences from the involucrin gene. A DNA fragment containing3.7 kb of the involucrin promoter directs transgene expression to thesuprabasal layers of the epidermis. This promoter fragment is amplifiedfrom total genomic DNA by PCR using primers to the known INV promotersequence (Lopez-Bayghen et al., J. Biol. Chem., 271(1): 512-520 (1996)).The Klf4 cDNA is cloned into a plasmid containing this involucrinpromoter fragment and used to generate stable cell lines of NIKS thatcontain this transgene. Stable cell lines are selected byco-transfecting NIKS cells with the INV/Klf4 plasmid and a plasmidexpressing the blasticidin resistance gene and growing the transfectedcells in the presence of blasticidin. Multiple blasticidin-resistantcell lines will be isolated and examined by Northern blot for increasedKlf4 expression as compared to cells transfected only with theblasticidin-resistance plasmid. While the involucrin promoter has beenused successfully to direct expression of several transgenes to thedifferentiating epidermis, it is possible that the INV/KLF4 constructwill not be expressed to high enough levels or in the proper temporal orspatial pattern to have an effect on barrier function. If Klf4expression from the involucrin promoter construct is not readilydetected, expression constructs are generated containing the promoterregions of another keratinocyte-specific gene, transglutaminase 3. TheKlf4 cDNA is cloned into an expression plasmid containing promotersequences from the transglutaminase 3 (TG3) gene. A TG3 promoterfragment containing 126 bp upstream and 10 bp downstream from thetranscription start site directs transgene expression to epithelialcells (Lee et al., J Biol Chem, 271(8): 4561-8 [1996]).

The Klf4 cDNA is cloned into a plasmid containing this TG3 promoterfragment and generate stable cell lines of NIKS that contain thistransgene. Stable cell lines are selected by co-transfecting NIKS cellswith the TG3/Klf4 plasmid and a plasmid expressing the blasticidinresistance gene and growing the transfected cells in the presence ofblasticidin. Multiple blasticidin-resistant cell lines are isolated andexamined by Northern blot for increased Klf4 expression as compared tocells transfected only with the blasticidin-resistance plasmid.

Stable NIKS cell lines that express Klf4 from the involucrin promoter orthe doxycycline-inducible system are examined in organotypic culture toconfirm that Klf4 is expressed under these culture conditions. Standardmedia and procedures for organotypic cultures are described inExample 1. Klf4-expressing NIKS cells are seeded onto a contractedcollagen matrix containing fibroblasts and grown in submerged culturefor 4 days before being lifted to the air interface. Organotypiccultures are fed cornification medium every 3 days and maintained at theair/liquid interface for 14 days to form a stratified epithelium.Cultures with NIKS expressing Klf4 from the inducible promoter are grownin media containing 1 microgram/ml doxycycline. Total RNA is isolatedfrom organotypic cultures by homogenizing the epidermal layer in Trizolreagent, extracting the homogenate with chloroform, and precipitatingtotal RNA with isopropanol. RNA is examined for Klf4 expression asdescribed above for monolayer cultures.

The effects of Klf4 expression on barrier function are examined bysurface capacitance measurement, lipid composition and ultrastructure ofthe organotypic cultures by the methods described in Example 1. Inaddition, some of the agents to be added to the cultures in Example 1,especially the PPAR and FXAR activators, may serve to activate otherregulatory genes that act in concert with Klf4. This hypothesis issupported by the ability of these agents to accelerate the in uterodevelopment of barrier function.

Data on the timing and extent of Klf4 expression using the twoexpression systems described herein will allow for the design ofstrategies to enhance barrier function by regulating Klf4 expression inNIKS organotypic cultures. The NIKS organotypic cultures have beenextensively characterized for ultrastructure and expression of keydifferentiation markers. Examination of these phenotypic properties ofthe culture in the presence of added Klf4 expression will provideadditional clues to the consequences of Klf4 expression.

Example 3 Lipid Content of Skin Equivalents

This example describes the preparation of skin equivalents withoptimized serum-free medium and a second set of skin equivalentsprepared with sub-optimal medium. Subsequently, the lipid content of theresulting cultures was determined.

Organotypic cultures were initiated by plating 350,000 NIKS cells ontodermal equivalents previously prepared within a 10 mm MILLICELL insert.The media used to complete this step was comprised of a base medium [3:1mixture of Ham's F12 medium/Dulbecco's modified Eagle's medium (DME),supplemented with 24 μg/ml adenine, 8.3 ng/ml cholera toxin, 5 μg/mlinsulin, 0.4 μg/ml hydrocortisone, with the final calcium concentrationadjusted to 1.88 mM] supplemented with 0.2% Fetal Clone II (a calf serumsubstitute).

Two days post-plating, the organotypic cultures were supplied with freshmedium to maintain growth. Cultures were supplied with either basemedium supplemented with 0.2% Fetal Clone II or base medium supplementedwith 0.2% Fetal Clone II and additional constituents (1 mg/mlendotoxin-free BSA, 1 ng/ml epidermal growth factor, 50 μg/ml ascorbicacid, 1 μM isoproterenol, 10 μM carnitine, 10 μM serine, 25 μM oleicacid, 15 μM linoleic acid, 7 μM arachidonic acid and 1 μM α-tocopherol).

Four days post-lifting, and every other day for the remainder of theculture period, the cultures were supplied with optimal medium (basemedium supplemented with 1 mg/ml endotoxin-free BSA, 1 ng/ml epidermalgrowth factor, 50 μg/ml ascorbic acid, 1 μM isoproterenol. 10 μMcarnitine, 10 μM serine, 25 μM oleic acid, 15 μM linoleic acid, 7 μMarachidonic acid and 1 μM α-tocopherol) or a sub-optimal medium (basemedium supplemented with 1 mg/ml endotoxin-free BSA, 1 ng/ml epidermalgrowth factor, 10 μM carnitine and 10 μM serine).

At the completion of the culture period, total lipids were extractedfrom the cultures and resolved by high-performance thin-layerchromatography (HPTLC). Following separation, the plates were charredand the resulting chromatograms were scanned by densitometry to quantifyindividual lipid species. The cultures grown in optimized culture mediumcontained a higher percentage of total ceramides than cultures grown insub-optimal medium (Table 3). In addition, the cultures grown in optimalmedium contained much higher levels of the polar ceramides 3, 4, 5, and6 than cultures grown under sub-optimal conditions.

TABLE 3 Ceramide content of skin cultures Optimal media Sub-optimalmedia Cer 6II 0.07% Cer 6I 0.41% 0.03% Cer 4/5 1.31% 0.46% Cer 3 1.37%0.44% Cer 2 2.09% 1.69% Cer 1 1.33% 0.42% Total ceramide 6.52% 3.11%

Example 4 Expression of GKLF in NIKS Cells

This example describes the expression in NIKS cells of GKLF, a proteinthought to mediate barrier function development in mice.

DNA (e.g., SEQ ID NO:2) encoding the GKLF protein was isolated by PCRand cloned into an expression vector containing the human involucrinpromoter. After verification of the GKLF and involucrin fragments by DNAsequencing, the constructs were introduced into NIKS cells bytransfection. Twenty-four hours after transfection, total RNA wasisolated from the transfected cells and expression of GKLF in thesecells was examined by reverse-transcription/PCR (RT-PCR).

A PCR product corresponding to spliced GKLF mRNA was detected in RNAfrom cells transfected with the involucrin/GKLF construct, but not incontrol RNA from cells transfected with empty vector. In addition, theGKLF PCR product was not detected in reactions from which reversetranscriptase was omitted. These results demonstrate that GKLF mRNA wasexpressed in transfected NIKS cells.

In a second set of experiments, DNA encoding GKLF was cloned into thepTRE2 vector, which allows for inducible expression of GKLF followingaddition of doxycycline. After verification of the GKLF and involucrinfragments by DNA sequencing, the constructs were introduced into NIKScells by transfection. Eight hours after transfection, doxycycline wasadded to half of the transfected cultures and all cultures wereincubated for an additional 16 hours. Twenty-four hours aftertransfection, total RNA was isolated from the transfected cells andexpression of GKLF in these cells was examined byreverse-transcription/PCR (RT-PCR). A PCR product corresponding tospliced GKLF mRNA was observed in samples with and without doxycycline,but more product was seen in samples following doxycycline addition. NoPCR products were detected in reactions from which reverse transcriptasewas omitted. These results demonstrate inducible expression of GKLF mRNAin transfected NIKS cells.

Example 5 Culture Methods

This example describes culture methods common to Examples 6-10.

Media. The organotypic culture process uses six different culture media:3T3 feeder cell medium (TM); fibroblast growth medium (FM); NIKS medium(NM); plating medium (PM); stratification medium A (SMA); andstratification medium B (SMB). TM is used to propagate 3T3 cells thatact as feeder cells for NIKS cells in monolayer culture. TM is a mixtureof Dulbecco's modified Eagle's medium (DME, GibcoBRL) supplemented with10% calf serum (Hyclone). FM is a mixture of Ham's F-12 medium(GibcoBRL) and 10% Fetal Clone II (Hyclone) serum. NM is used to growNIKS keratinocytes. NM is a 3:1 mixture of Ham's F-12 medium (GibcoBRL)and DME supplemented with 2.5% Fetal Clone II (Hyclone), 0.4 μg/mlhydrocortisone (Calbiochem), 8.4 ng/ml cholera toxin (ICN), 5 μg/mlinsulin (GibcoBRL), 24 μg/ml adenine (Sigma) and 10 ng/ml epidermalgrowth factor (EGF, R&D systems). PM is the medium used when NIKS cellsare seeded onto a dermal equivalent. PM is the same as NM except thatEGF is removed, CaCl₂ (Sigma) is supplemented to a final calciumconcentration of 1.88 mM, and only 0.2% Fetal Clone II serum is added.SMA is the same as PM with the addition of 1 mg/ml bovine serum albumin(BSA), 1 μM isoproterenol, 10 μM carnitine, 10 μM serine, 25 μM oleicacid, 15 μM linoleic acid, 7 μM arachidonic acid, 1 μM α-tocopherol,0.05 mg/ml ascorbic acid (all from Sigma), and 1 ng/ml EGF. SMB is usedduring the epidermal stratification phase of STRATATEST skin equivalentand STRATAGRAFT skin equivalent growth. SMB is the same as SMA butwithout the presence of the Fetal Clone II serum supplement.

Feeder preparation. Prior to starting STRATATEST skin equivalent orSTRATAGRAFT skin equivalent organotypic cultures, 3T3 feeder cells areprepared and then used either fresh or frozen for later use. 3T3 cellsare grown to confluence and treated with mitomycin-C (4 ug/ml ofmitomycin-C in TM, Roche) for two hours. The cells are then washed,resuspended, and plated at a density of 1.25×10⁶ per 100 mm tissueculture dish to support NIKS growth. If frozen feeders are used, asingle frozen ampoule containing 1 ml with 2.5×10⁶ is thawed, dilutedwith fresh TM and plated onto one or more 100 mm tissue culture dishes.This is done for as many dishes as will be needed for NIKS cell growthone day prior to plating the NIKS cells.

Dermal equivalent preparation. Frozen NHDF cells are thawed and plated.The cells are fed FM the next day to remove residual cryoprotectant andsubsequently to maintain cell growth. Preconfluent NHDF cells areharvested for use in the dermal equivalent. To prepare the dermalequivalent, rat tail tendon collagen (Type I, Becton-Dickinson) is firstdiluted to 3 mg/ml in 0.03N acetic acid and chilled on ice. A mixture ofconcentrated Ham's F12 medium (8.7× normal strength, buffered with HEPESat pH 7.5) is mixed with Fetal Clone II. These two solutions are 11.5and 10% of the final solution volume. 1 N NaOH is added to the mediummixture (2.5% of final solution). The diluted collagen (74%) is thenadded to the mixture. A 2% volume of suspended fibroblasts (2.5×10⁶cells/ml for the dermal equivalent of STRATATEST and 1×10⁶ for dermalequivalent of STRATAGRAFT) is added to the mixture. The solution ismixed gently but thoroughly. 100 μl is aliquoted into tissue cultureinserts (MILLICELL from Millipore Corp.) placed 25 in a 100 mm tissueculture dish for STRATATEST. The STRATAGRAFT skin equivalent usesTRANSWELL inserts from Coming. A 13 ml dermal equivalent is poured intoeach insert making it roughly three times the thickness of a STRATATESTdermal equivalent. After 30 minutes for gel formation, the dishcontaining STRATATEST dermal equivalents is flooded with 20 ml of FM.One or two drops FM are placed on the surface of each STRATATEST dermalequivalent. For STRATAGRAFT dermal equivalents, 80 ml of FM is placedaround the TRANSWELL insert in a 150 mm tissue culture dish and 10 ml isplaced on top of the dermal equivalent. The inserts are placed in 37°C., 5% CO₂, 90% relative humidity incubator until used. One day prior toseeding the dermal equivalents with NIKS cells, they are lifted to theair interface by placing them onto a sterile stainless steel mesh withtwo wicking pads (S&S Biopath) on top to supply medium through thebottom of the tissue culture insert.

NIKS Growth and Seeding. Feeders are prepared fresh or thawed and platedin TM one day prior to NIKS plating. NIKS cells are plated onto thefeeders at a density of approximately 3×10⁵ cells per 100 mm dish. Ifthe NIKS cells are newly thawed, they are fed fresh NM one daypost-plating to remove residual cryoprotectant. The NIKS cells are fedNM to maintain growth as required When cell approach confluence, theNIKS cells are harvested, counted, and resuspended in PM. 4.65×10⁵ NIKScells/cm² are seeded onto the surface of the MIILLICELL or TRANSWELLdermal equivalents, which have been lifted to the air interface for oneday. The dishes are fed PM to flood underneath the metal lifter andplaced back into the incubator. Two days later, the cultures are fedSMA. After an additional two days, the cultures are fed SMB andtransferred to a 75% humidity incubator where they remain, maintainedwith additional SMB feedings, until mature.

Example 6

This example describes the preparation of dermal equivalents using 1mg/ml collagen. Briefly, 24 ml Ham's F12 medium prepared at 10×concentration was mixed with 4.8 ml sterile H₂O, 2.4 mlPenicillin/Streptomycin mixture and 24 ml Fetal Clone II in a 50 mlconical tube. Rat tail tendon collagen Type I (1.46 ml) at 4.11 mg/mlwas diluted with 1.882 ml sterile H₂O and 2.658 ml of 0.05% acetic acid.Normal human dermal fibroblasts were harvested from culture andresuspended at a cell density of 10⁶ and 10⁴ cells/ml. 0.815 ml of themedium-containing mixture was combined with 2.619 ml of diluted collagenand 34 μl of fibroblasts at 10⁴ cells/ml. 116.5 μl of this mixture wasaliquotted into tissue culture inserts (25 of which are in a Petri dish)and allowed to gel for 15 minutes at 37° C. An additional 0.815 ml ofthe medium-containing mixture was combined with another 2.619 ml ofdiluted collagen and 137 μl of fibroblasts at 10⁶ cells/ml. 116.5 μl ofthis mixture was aliquotted into the tissue culture inserts on top ofthe previous gel and allowed to gel for 30 minutes.

The petri dish was then flooded with 20 ml of FM medium and incubatedfor 5 days. The FM was then removed and the liquid aspirated from thesurfaces of the dermal equivalents. NIKS cells were harvested usingstandard procedures, resuspended at 2.345×10⁶ cells/ml in plating medium(PM). 150 μl of this suspension was put on the surface of each dermalequivalent and allowed to incubate for 2 hours. The seeded dermalequivalents were then flooded with 20 ml PM. After two days thesubmerged cultures were refed with PM.

After two more days the medium was removed from the petri dish as wellas from the surface of the cultures. The cultures were lifted to the airinterface and fed approximately 30 ml of PM supplemented to 2% serumevery 2 days. Cultures were analyzed 14 days after they were seeded.None of the cultures had complete epidermal coverage of the dermalequivalent. Thus they were unsuitable for commercial application.

Example 7

This example describes the preparation of dermal equivalents using 3mg/ml collagen. 4.785 ml Ham's F12 medium prepared at 10× concentrationwas mixed with 0.946 ml sterile H₂O, 0.473 ml Penicillin/Streptomycinmixture, and 4.785 ml Fetal Clone II in a 50 ml conical tube. 4.6 ml ofthis medium mixture was mixed with 0.242 ml sterile H₂O and 0.289 ml 1NNaOH. 0.92 ml of the mixture was mixed with 3 ml rat tail tendoncollagen Type I at 3.11 mg/ml. To this was added 186 μl of a humandermal fibroblast suspension at 10⁶ cells/ml. 100 μl of this mixture wasplaced into the MILLICELL inserts (1 cm diam) and allowed to gel for 30minutes. The petri dish was then flooded with 20 ml of FM medium andallowed to incubate. After 5 days, the FM was removed and the liquidaspirated from the surfaces of the dermal equivalents. NIKS™ cells wereharvested using standard procedures, resuspended at 2.345×10⁶ cells/mlin plating medium (PM). 150 μl of this suspension was put on the surfaceof each dermis and allowed to incubate for 2 hours. The seeded dermalequivalents were then flooded with 20 ml PM. After two more days themedium was removed from the petri dish (including the surface of thecultures) and the cultures were lifted to the air interface and fedapproximately 30 ml of stratification medium every 2 days. Cultures wereanalyzed 14 days after they were seeded. At the completion of culturegrowth, all of the cultures had complete coverage of the dermalequivalent with epidermis and were smooth and dry in appearance. Thusthey were highly acceptable for commercial application.

Example 8

This example demonstrates the beneficial effects of prelifting thedermal equivalent prior to seeding with keratinocytes. 1.31 ml Ham's F12medium prepared at 10× concentration was mixed with 0.328 ml sterileH₂O, 0.148 ml Penicillin/Streptomycin mixture, and 1.472 ml Fetal CloneII in a 50 ml conical tube and 1.63 ml (˜half) was split into a secondtube. 2.92 ml of rat tail tendon collagen (4.11 mg/ml) was mixed with3.764 ml sterile H₂O, and 5.316 ml of 0.05% acetic acid to give 1 mg/mlcollagen in 0.05% acetic acid. 5.24 ml of the diluted collagen was addedto 1.63 ml of the medium mixture. 74 μl of human dermal fibroblast cellsharvested with standard protocols at a cell density of 10⁴ cells/ml wasadded and gently mixed. 116.5 μl of this mixture was aliquotted intotissue culture inserts (25 to a Petri dish) and allowed to gel for 15minutes at 37° C. Another 5.24 ml of collagen was added to the second1.63 ml of medium mixture along with 274 μl of fibroblasts at 10⁶cells/ml. 116.5 μl was added to each insert on top of the first gelledcollagen layer. This was allowed to gel for 30 minutes at 37° C. Thepetri dishes were then flooded with 20 ml of FM so that the dermalequivalents could mature submerged in medium. After four days the mediumwas removed from the petri dish (including from the surface of thecultures) and the cultures were lifted to the air interface and fedapproximately 30 ml of FM. The cultures were left in the incubator inthis state overnight. Then they were seeded with 150 μl of NIKSkeratinocytes harvested from monolayer culture using standard protocolsat a cell density of 2.345×10⁶ cells/ml. After seeding, the cultureswere fed PM and returned to the incubator. Two days later, the cultureswere fed with SMA, and every second day thereafter cultures were fedwith SMB for a total of eight feedings. At the completion of culturegrowth, all of the cultures had complete coverage of the dermalequivalent with epidermis and were smooth and dry in appearance.Histology revealed that the prelifted samples had approximately equalthicknesses of dermis and epidermis, and all stratified layers werepresent in the epidermis.

Example 9

This example describes the effect of prelifting for the entire life ofthe dermal equivalent. Cultures were prepared exactly as in thesuccessful experiment listed above, with the exception that they werenever submerged. The gels were poured with the MILLICELL inserts liftedto the air interface and all subsequent seeding and feeding took placewith the cultures lifted. At the completion of culture growth, one inten of the cultures had complete coverage of the dermal equivalent withepidermis. This effect is apparently due in part to poor adherence ofthe dermal equivalent to the bottom of the MILLICELL insert since thedermal equivalent had pulled away from the sides in most samples.Histology indicated that the dermal and epidermal layer thicknesses werehighly variable. Likewise epidermal stratification ranged from welldifferentiated to only monolayer coverage which was unacceptable forcommercial use.

Example 10

This example describes the optimization of a serum-free media thatsupports full stratification of keratinocytes in organotypic culturethat also results in skin equivalents with improved barrier function.

Organotypic cultures were initiated by plating 350,000 NIKS cells onto adermal equivalent previously prepared within a 10 mm MILLICELL insert.The media used to complete this step was comprised of a base medium (3:1mixture of Ham's F12 medium/Dulbecco's modified Eagle's medium (DME),supplemented with 24 μg/ml adenine, 8.3 ng/ml cholera toxin, 5 μg/mlinsulin, 0.4 μg/ml hydrocortisone, 100 units/ml penicillin, 100 μg/mlstreptomycin, with final calcium concentration adjusted to 1.88 mMthrough the addition of CaCl₂) supplemented with 0.2% Fetal Clone II.

Two days post-plating, the organotypic cultures were supplied with freshmedia to maintain growth. Cultures were supplied with either base mediasupplemented with 0.2% Fetal Clone II or base media with additionalconstituents (1 mg/ml low endotoxin bovine serum albumin, 1 ng/mlepidermal growth factor, 1 μM isoproterenol, 10 μM carnitine, 10 μMserine, 25 μM oleic acid, 15 μM linoleic acid, 7 μM arachidonic acid, 1μM α-tocopherol, and 0.05 mg/ml ascorbic acid) supplemented with 0.2%Fetal Clone II.

Four days post-plating, and for the remainder of the experiment, theorganotypic cultures were supplied with one of six media formulations.Cultures that had previously received base media supplemented with 0.2%Fetal Clone II, were supplied with either base media without Fetal CloneII supplementation, or base media supplemented with 0.2% Fetal Clone II,or base media supplemented with 2% Fetal Clone II. Cultures that hadpreviously received base media with additional constituents supplementedwith 0.2% Fetal Clone II, were supplied with either base media withadditional constituents without Fetal Clone II supplementation, or basemedia with additional constituents supplemented with 0.2% Fetal CloneII, or base media with additional constituents supplemented with 2%Fetal Clone II.

Three criteria were used to evaluate the impact of the mediaformulations. Visual inspection was used to determine the extent ofcontiguous cellular surface coverage. Impedance meter readings were usedto measure the resulting barrier function of cultures. Viability oftissue post-exposure to 0.1% SDS was used as a practical evaluation ofbarrier function. For all criteria, organotypic cultures maintained inbase media with additional constituents performed better than base mediawithout additional constituents. The exclusion of serum did not hinderthe performance of organotypic cultures as long as additionalconstituents were supplied.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology, genetics, or related fields are intended to be withinthe scope of the following claims.

1. A method of making a skin equivalent having improved barrierfunction, the method comprising: providing (a) a composition comprisingnear-diploid immortalized keratinocyte (NIKS) cells and (b) a culturemedium comprising ascorbic acid, linoleic acid, isoproterenol,carnitine, serine, oleic acid, arachidonic acid, α-tocopherol, andepidermal growth factor in amounts effective to improve barrierfunction; and culturing said composition (a) in said culture medium (b)under conditions wherein said culturing provides a skin equivalenthaving a surface electrical capacitance of 40 to 240 pF measured over a10 second interval in vitro.
 2. The method of claim 1, wherein saidascorbic acid is at a concentration of about 10 to 100 micrograms/ml. 3.The method of claim 1, wherein said ascorbic acid is at a concentrationof about 0.05 mg/ml.
 4. The method of claim 1, wherein said linoleicacid is at a concentration of about 5 to 80 micromolar.
 5. The method ofclaim 1, wherein in said skin equivalent the content of ceramides 5, 6,and 7is about 20 to about 50% of the total ceramide content.
 6. Themethod of claim 1, wherein in said skin equivalent the content ofceramide 2 is about 10 to about 40% of the total ceramide content. 7.The skin equivalent produced by the method of claim 1.